Mineral coated scaffolds

ABSTRACT

Provided is a composition for a scaffold having a mineral coating similar to bone. Also provided is a method for mineral coating a scaffold so as to promote mineral coating of the scaffold with a plate-like nanostructure and a carbonate-substituted, calcium-deficient hydroxyapatite phase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a: (1) U.S. Continuation-In-Part of U.S.Nonprovisional application Ser. No. 13/036,470 filed 28 Feb. 2011, whichis a Continuation-In-Part of U.S. Nonprovisional application Ser. No.11/927,322 filed 29 Oct. 2007, which claims the benefit of U.S.Provisional Application No. 60/855,235 filed 30 Oct. 2006; (2) U.S.Continuation-In-Part of U.S. National Phase application Ser. No.13/879,178 filed 18 Feb. 2014, which is a 371 of InternationalApplication No. PCT/US09/58419 filed 25 Sep. 2009, which claims thebenefit of U.S. Provisional Application No. 61/100,062 filed 25 Sep.2008; and (3) U.S. Continuation-In-Part of U.S. Nonprovisionalapplication Ser. No. 13/407,441 filed 28 Feb. 2012, which claimspriority to U.S. Provisional Application No. 61/447,352 filed 28 Feb.2011.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant R03AR052893awarded by The National Institutes of Health. The government has certainrights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

Not Applicable.

FIELD OF THE INVENTION

The present disclosure generally relates to compositions and methods formineral coated scaffolds.

BACKGROUND OF THE INVENTION

Hybrid materials composed of organic polymers coated with inorganicminerals have attracted much attention in biology and medicine due totheir combination of advantageous properties. Polymeric materials can bea desirable base material for biomedical applications, as they can beprocessed into a variety of sizes and geometries, and can be designed tobioresorb in a controllable timeframe. Therefore, polymeric biomaterialshave been featured in a variety of applications, including medicaldevices, tissue engineering scaffolds, and drug delivery systems.

Calcium phosphate based mineral coatings represent desirable surfacesfor biomedical applications, as they can be similar in composition tobone tissue, and have been shown to promote favorable interactions withnatural bone, a property termed “bioactivity”. For example,hydroxyapatite, the major inorganic component of bone mineral, isosteoconductive (Ducheyne et al., 1999), and may also be capable ofinducing new bone formation in vivo (Habibovic et al., 2006).

A particular subset of approaches used to grow hydroxyapatite coatingson biomaterials surfaces mimics some aspects of naturalbiomineralization processes, and has therefore been termed “biomimetic”or “bioinspired” (Hong et al., 2006; Gao and Koumoto, 2005; Leveque etal., 2004; Green et al., 2006). This type of approach is a practicallyand economically attractive alternative to high-temperature commercialprocessing methods such as plasma-spraying (Gledhill et al., 2001),sputter coating (Yamashita et al., 1994), and laser deposition(Fernandez-Pradas et al., 1998). Kokubo et al. first reportedbioinspired growth of apatite coatings on bioactive CaO—SiO₂ glass in asimulated body fluid (SBF), which had ion concentrations nearly equal tothose of human blood plasma and was held at physiologic temperature andpH (Kokubo et al., 1990). A series of subsequent studies reportedmineral growth using novel formulations of SBF (Oyane et al., 2003),variation in the mineral growth process (Miyaji et al., 1999), orvariations in the base materials (Yogogawa et al., 1997). The basis formineral nucleation in these studies involved interactions of mineralions in solution with polar functional groups on the materials surface,such as Si—OH (Li et al., 1992), Ti—OH (Barrere et al., 2004) and Zr—OH(Uchida et al., 2001). A series of recent studies has extended thebioinspired mineralization process to include formation of a bone-likehydroxyapatite coating on biodegradable polymer films (Murphy andMooney, 2002) or porous scaffolds (Murphy et al., 2000; Zhang and Ma,2004; Bajpai and Singh, 2007). The mechanism for mineral nucleation andgrowth on these materials is based on the interaction of carboxylate andhydroxyl groups on the hydrolyzed surface with calcium- andphosphate-rich nuclei in solution, creating a driving force forheterogeneous nucleation and mineral growth (Murphy and Mooney, 2002).This coating process is particularly suitable for biocompatible implantsand biodegradable polymers, as it can be carried out at physiologicaltemperature and pH (Tanahashi et al., 1994), and the mild processingconditions also suggest that it is possible to incorporate biologicallyactive molecules such as polypeptides and polynucleotides, during thecoating process.

Previous studies have shown that demineralized bone matrix (DBM) is anosteogenic material, but Ozturk et al. 2006 Int Orth. 30, 147-152, showsthat DBM alone shows better osteoconductive properties than theDBM/hydroxyapatite (HA) mixture.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofa mineral coated scaffold that has or can include a mineral coatingsimilar to bone.

One aspect of the present disclosure provides a method for producing amineral coated scaffold. In some embodiments, the method includescontacting a scaffold comprising a matrix material and a modifiedsimulated body fluid. In some embodiments, the method includesincubating the scaffold and the modified simulated body fluid for aperiod of time under conditions sufficient to form a mineral coatedscaffold.

In some embodiments, the method includes a biodegradable matrixmaterial. In some embodiments, the matrix material comprisespolycaprolactone (PCL), polyetheretherketone (PEEK), or titanium (Ti).

In some embodiments, the method includes forming a modified simulatedbody fluid comprising combining NaCl, KCl, MgCl₂, MgSO₄, NaHCO₃, CaCl₂,and KH₂PO₄ to form the modified simulated body fluid. In someembodiments, the method includes combining NaCl at a concentration ofabout 100 mM to about 200 mM; KCl at a concentration of about 1 mM toabout 8 mM; MgCl₂ at a concentration of about 0.2 mM to about 5 mM;MgSO₄ at a concentration of about 0.2 mM to about 5 mM; NaHCO₃ at aconcentration of about 1 mM to about 100 mM; CaCl₂ at a concentration ofabout 2 mM to about 20 mM; and KH₂PO₄ at a concentration of about 0.5 mMto about 10 mM.

In some embodiments, the method includes forming a modified simulatedbody fluid that comprises combining NaCl at a concentration of about 141mM; KCl at a concentration of about 4.0 mM; MgCl₂ at a concentration ofabout 1.0 mM; MgSO₄ at a concentration of about 0.5 mM; NaHCO₃ at aconcentration of about 4.2 mM; CaCl₂ at a concentration of about 5 mM;and KH₂PO₄ at a concentration of about 2.0 mM.

In some embodiments, the method includes a modified simulated body fluidcomprising a buffer. In some embodiments, the method includes a modifiedsimulated body fluid comprising a buffer at a concentration of about 20mM. In some embodiments, the method includes a modified simulated bodyfluid comprising a buffer selected from the group consisting of DPBS,Tris, Tris-HCl, Tris-buffered saline, or PBS.

In some embodiments, the method includes incubating the scaffold and themodified simulated body fluid for about 1 day to about 21 days. In someembodiments, the method includes incubating the scaffold and themodified simulated body fluid for about 7 days; at least about 8 days;at least about 9 days; at least about 10 days; at least about 11 days;at least about 12 days; at least about 13 days; or at least about 14days. In some embodiments, the method includes incubating the scaffoldand the modified simulated body fluid for about 5 days to about 14 days.

In some embodiments, the method includes hydrolyzing the scaffold. Insome embodiments, the method includes roughening the scaffold.

In some embodiments, the method includes incubation comprising heatingthe modified simulated body fluid to physiologic temperature oradjusting to a physiologic pH. In some embodiments, the method includesincubating at physiologic temperature of about 37° C. or physiologicalpH of about 6.8.

In some embodiments, the method includes incubating comprising replacingthe modified simulated body fluid, replenishing the modified simulatedbody fluid, removing the modified simulated body fluid, or adding themodified simulated body fluid, wherein incubating comprises maintaininga concentration of modified simulated body fluid. In some embodiments,the method includes maintaining the concentration of modified simulatedbody fluid comprising replacing, replenishing, removing, or addingmodified simulated body fluid, NaCl, KCl, MgCl₂, MgSO₄, NaHCO₃, CaCl₂,or KH₂PO₄, or a combination thereof.

In some embodiments, the method includes the coating comprising (i)about 9% to about 100% hydroxyapatite; (ii) about 90% to about 100%hydroxyapatite; or (iii) about 97% hydroxyapatite.

In some embodiments, the method includes the coating comprising (i)about 0% to about 30% octacalcium phosphate; (ii) about 0% to about 3%octacalcium phosphate; or (iii) about 3% octacalcium phosphate.

In some embodiments, the method includes the coating comprising (i)between about 2% and about 100% porosity; or (ii) between about 20% andabout 28% porosity.

In some embodiments, the method includes the coating comprising (i)between about 1 nm and about 3500 nm pore diameter; or (ii) betweenabout 100 nm and about 350 nm pore diameter.

In some embodiments, the method includes the scaffold comprising (i)between about 200 μm and about 525 μm pore diameter; or (ii) betweenabout 25 μm to about 65 μm pore diameter.

In some embodiments, the method includes the coating comprising (i)about 0.1 to about 18 Ca/P; or (ii) about 1.1 to about 1.76 Ca/P(calcium to phosphate ratio).

In some embodiments, the method includes the coating comprising (i)about 1.67 to about 1.76 Ca/P; (ii) about 1.1 to about 1.3 Ca/P; or(iii) about 1.37 to about 1.61 Ca/P.

In some embodiments, the method includes the coating comprising (i)about 9% to about 100% crystallinity; (ii) about 90% to about 100%crystallinity; or (iii) about 96.5% crystallinity.

In some embodiments, the method includes the coating comprising silverparticles and/or demineralized bone matrix (DBM).

In some embodiments, the method includes lyophilizing the coatedscaffold.

Another aspect provides a mineral coated scaffold comprising a matrixmaterial, wherein the mineral coating of the scaffold comprises aplate-like nanostructure and a carbonate-substituted, calcium-deficienthydroxyapatite component.

Another aspect provides a mineral coated scaffold comprising a matrixmaterial, wherein the mineral coating comprises a plate-likenanostructure and a carbonate-substituted, calcium-deficienthydroxyapatite component.

In some embodiments, the coating comprises about 9% to about 100%hydroxyapatite; about 90% to about 100% hydroxyapatite; or about 97%hydroxyapatite; about 0% to about 30% octacalcium phosphate; about 0% toabout 3% octacalcium phosphate; or about 3% octacalcium phosphate;between about 2% and about 100% porosity; or between about 20% and about28% porosity; between about 1 nm and about 3500 nm pore diameter; orbetween about 100 nm and about 350 nm pore diameter; about 0.1 to about18 Ca/P; about 1.1 to about 1.76 Ca/P; about 1.67 to about 1.76 Ca/P;about 1.1 to about 1.3 Ca/P; or about 1.37 to about 1.61 Ca/P; or about9% to about 100% crystallinity; about 90% to about 100% crystallinity;or about 96.5% crystallinity.

In some embodiments, the scaffold comprises silver particles and/ordemineralized bone matrix (DBM).

In some embodiments, the scaffold exhibits improved osteoinductiveproperties compared to the mineral coated scaffold without the DBM.

In some embodiments, the scaffold comprises silver particles in aneffective amount to provide antimicrobial, antibacterial, biostatic, oranti-infection properties.

In some embodiments the silver particles are provided as silvernanoparticles or silver microparticles.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A-1B are a series of micrographs depicting the coating surfaces ona PCL scaffold.

FIG. 1A shows a representative SEM micrograph of the coating surfaces onPCL scaffold at low magnification.

FIG. 1B shows a representative SEM micrograph of the coating surfaces onPCL scaffold at high magnification.

FIG. 2A-2F are a series of micrographs depicting the coating surfaces ona PCL scaffold after incubation with DPBS.

FIG. 2A shows SEM micrographs of the coating surfaces after incubatingin DPBS on day 3.

FIG. 2B shows SEM micrographs of the coating surfaces after incubatingin DPBS on day 7.

FIG. 2C shows SEM micrographs of the coating surfaces after incubatingin DPBS on day 14.

FIG. 2D shows SEM micrographs of the coating surfaces after incubatingin Tris-HCl on day 3.

FIG. 2E shows SEM micrographs of the coating surfaces after incubatingin Tris-HCl on day 7.

FIG. 2F shows SEM micrographs of the coating surfaces after incubatingin Tris-HCl on day 14.

FIG. 3A-3B are a series of bar graphs depicting the amount of cumulativerelease of phosphate or calcium.

FIG. 3A shows the amount of cumulative phosphate release in Tris-HCl.

FIG. 3B shows the amount of cumulative calcium release in Tris-HCl andDPBS.

FIG. 4A-4B are a series of micrographs depicting the surfaces on atitanium (Ti) scaffold.

FIG. 4A shows SEM micrographs of the uncoated Ti.

FIG. 4B shows SEM micrographs of the coated Ti.

FIG. 5A-5B are a series of micrographs depicting the uncoated surfaceson a Ti scaffold.

FIG. 5A shows SEM micrographs of the uncoated Ti (scale bar=1 mm).

FIG. 5B shows SEM micrographs of the uncoated Ti (scale bar=500 μm).

FIG. 5C shows SEM micrographs of the uncoated Ti (scale bar=200 μm).

FIG. 5D shows SEM micrographs of the uncoated Ti (scale bar=50 μm).

FIG. 6A-6D are a series of micrographs depicting the coated surfaces ona Ti scaffold.

FIG. 6A shows SEM micrographs of the coated Ti (scale bar=1 mm).

FIG. 6B shows SEM micrographs of the coated Ti (scale bar=500 μm).

FIG. 6C shows SEM micrographs of the coated Ti (scale bar=200 μm).

FIG. 6D shows SEM micrographs of the coated Ti (scale bar=50 μm).

FIG. 7A shows SEM micrographs of the coated Ti (scale bar=20 μm).

FIG. 7B shows SEM micrographs of the coated Ti (scale bar=10 μm).

FIG. 8 shows the coating composition on Ti is consistent with thecoating on a PCL surface.

FIG. 9 shows the coating thickness on Ti is in the range of 5-15 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery ofadvantageous properties of methods of producing mineral coated scaffoldsto form a coating similar is structure and composition to bone. As shownherein, the coated scaffolds have been produced and characterized toprovide advantageous properties.

Scaffold

One aspect of the present disclosure provides a scaffold suitable formineral coating. A scaffold can provide a substrate for the growth ofbone mineral. The scaffold including a simulated body fluid can promotemineral coating of the scaffold with a plate-like nanostructure and acarbonate-substituted, calcium-deficient hydroxyapatite phase. Ascaffold can be as described in U.S. application Ser. Nos. 13/407,441;13/879,178; and 13/036,470 and are incorporated herein by reference.

The scaffold can be formed of any suitable material known in the art.The selection of the scaffold material for any particular applicationcan be made without undue experimentation. For example, an applicationof the scaffold can be for a medical device or an implant.

A scaffold can comprise a matrix material. As used herein, a “matrix”can be a material, e.g., a polymer, in which one or more ingredients canbe suspended. A “scaffold” is understood to have a secondary or tertiarystructure (e.g., a columnar structure or a porous structure in which oneor more ingredients can permeate). The present disclosure is not limitedto any particular matrix or scaffold. Preferably, the matrix or scaffoldis biodegradable.

As described herein, a scaffold can be a hydrolyzable scaffold. Forexample, the scaffold can be biodegradable. As another example, thescaffold can be a polycaprolactone (PCL) scaffold orpolyetheretherketone (PEEK) scaffold. As another example, the PCLscaffold can be hydrolyzed using NaOH.

As described herein, a scaffold can be coated. For example, the coatingcan be a mineral coating, as described herein.

A scaffold or matrix, as described herein, can include one or morecomponents fabricated in whole or in part from a polymer material, suchas a degradable polymer material, a porous polymer material, or adegradable porous polymer material. Suitable scaffold materials arediscussed in, for example, Ma and Elisseeff, ed. (2005) Scaffolding inTissue Engineering, CRC, ISBN 1574445219; Saltzman (2004) TissueEngineering: Engineering Principles for the Design of Replacement Organsand Tissues, Oxford ISBN 019514130X.

A scaffold made in whole or in part from a polymer material can: providestructural and/or functional features of the target tissue (e.g., bone);allow cell attachment and migration; deliver and retain cells andbiochemical factors; enable diffusion of cell nutrients and expressedproducts; or exert certain mechanical and biological influences tomodify the behavior of the cell phase.

Scaffold materials can be biocompatible materials that generally form aporous, microcellular matrix, which can provide a physical support or anadhesive substrate for introducing bioactive agents or cells duringfabrication, culturing, or in vivo implantation.

As described herein, a scaffold can be a metallic scaffold. As anotherexample the metallic scaffold can be a biocompatible metallic scaffold.As another example the metallic scaffold can be a titanium (Ti)scaffold. As another example the Ti scaffold can be a Ti alloy scaffold.As another example, a Ti alloy can be Ti-35Nb-5Ta-7Zr, or TNZT.

A scaffold or matrix, as described herein, can include one or morecomponents fabricated in whole or in part from a metallic material, suchas titanium or stainless steel. Suitable metallic scaffold materials arediscussed in, for example, Alvarez et al. 2009 Materials 2, 790-832.

As described herein, the metal scaffold can be treated with physical orchemical methods to impart roughness to the surface. As another example,the treatment can impart submicron- or nano-roughness to the metalsurface. As another example, the metal scaffold can be treated with analkaline solution. As another example, the alkaline solution can be NaOHor KOH. Suitable treatments for metallic scaffold materials arediscussed in, for example, Alvarez et al. 2009 Materials 2, 790-832.

As described herein, the roughened titanium can have larger porestructures and smaller pore structures.

As described wherein, the larger pore structures can be at least about200 μm. For example, the larger pore structure can be at least about 200μm; at least about 225 μm; at least about 250 μm; at least about 275 μm;at least about 300 μm; at least about 325 μm; at least about 350 μm; atleast about 375 μm; at least about 400 μm; at least about 425 μm; atleast about 450 μm; at least about 475 μm; at least about 500 μm; or atleast about 525 μm. It is understood that recitation of the abovediscrete values includes a range between each recited value.

As described wherein, the larger pore structures can be at about 200 μm;about 225 μm; about 250 μm; about 275 μm; about 300 μm; about 325 μm;about 350 μm; about 375 μm; about 400 μm; about 425 μm; about 450 μm;about 475 μm; about 500 μm; or about 525 μm. It is understood thatrecitation of the above discrete values includes a range between eachrecited value.

As described wherein, the smaller pore structures can be at least about200 μm. For example, the smaller pore structure can be at least about 10μm; at least about 15 μm; at least about 20 μm; at least about 25 μm; atleast about 30 μm; at least about 35 μm; at least about 40 μm; at leastabout 45 μm; at least about 50 μm; at least about 65 μm; at least about70 μm; at least about 75 μm; at least about 80 μm; at least about 85 μm;at least about 90 μm; at least about 95 μm; or at least about 100 μm. Itis understood that recitation of the above discrete values includes arange between each recited value.

As described wherein, the smaller pore structures can be about 10 μm;about 15 μm; about 20 μm; about 25 μm; about 30 μm; about 35 μm; about40 μm; about 45 μm; about 50 μm; about 55 μm; about 60 μm; about 65 μm;about 70 μm; about 75 μm; about 80 μm; about 85 μm; about 90 μm; about95 μm; or about 100 μm. It is understood that recitation of the abovediscrete values includes a range between each recited value.

As described herein, the average pore volume can be about 10%; about20%; about 30%; about 40%; about 50%; about 60%; about 70%; about 80%;or about 90%.

As described herein, the average surface roughness can be about 10 μm;20 μm; 30 μm; 40 μm, 50 μm; 60 μm; 70 μm; 80 μm; 90 μm; 100 μm; 110 μm;120 μm; 130 μm; 140 μm; 150 μm; 160 μm; 170 μm; 180 μm; 190 μm; or 200μm. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

Generally, a biocompatible material can be one which stimulates at mostonly a mild, often transient, implantation response, as opposed to asevere or escalating response. A biodegradable or degradable materialcan be generally understood to decompose under normal in vivophysiological conditions into components which can be metabolized orexcreted.

Material biodegradability can provide for absorption of the matrix bythe surrounding tissues and can eliminate the necessity of a surgicalremoval. The rate at which degradation occurs can coincide as much aspossible with the rate of mineral formation. Thus, while the mineralcoating is fabricating their own natural structure around themselves,the scaffold or components thereof can provide structural integrity andeventually break down leaving the mineral which can assume themechanical load. One or more scaffold materials can be modified so as toincrease biodegradability. For example, polycaprolactone (PCL) is abiodegradable polyester by hydrolysis of its ester linkages inphysiological conditions, and can be further modified with ring openingpolymerization to increase its biodegradability.

In some embodiments, the scaffold comprises a negative charge, which canpromote the deposition of the calcium containing material. The negativecharge can be provided by any moiety present on the scaffold, forexample a carboxylate group, as is present inpoly(D,L-lactide-co-glycolide) (PLG).

In some embodiments, the scaffolds provided herein can be formed frompolycaprolactone, a biocompatible and biodegradable polymer. However,other polymers are known to be biocompatible, and can be used for thescaffolds described herein. Nonlimiting examples of suitablebiodegradable materials include polycaprolactone, polylactide,polyglycolide, poly(lactide-glycolide), poly(propylene fumarate),poly(caprolactone fumarate), polyethylene glycol,poly(glycolide-co-caprolactone), polysaccharides (e.g. alginate),chitosan, polyphosphazene, polyacrylate, polyethyleneoxide-polypropylene glycol block copolymer, fibrin, collagen,fibronectin, polyvinylpyrrolidone, hyaluronic acid, polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates, polyurethanes, polyacrylates,ethylene-vinyl acetate polymers and other acyl substituted celluloseacetates and derivatives thereof, and analogs, mixtures, combinationsand derivatives of any of the above.

In some embodiments, a scaffold, or portion or component thereof,comprises a material having a porous microstructure. Pores of ascaffold, or portion or component thereof, can mimic internal bonestructure, allow adherence of cells, provide an open volume for seedingof cells, provide an open volume for growth factors or other additives,allow adherence of another matrix layer, serve as conduits forvascularization, provide internal bone features, or facilitateperfusion. A scaffold material with a high porosity and an adequate poresize is preferred so as to facilitate mineralization, cell introduction,and diffusion throughout the whole structure of both cells andnutrients.

Pores of a scaffold material can be engineered to be of variousdiameters. For example, the pores of a scaffold material can have adiameter range from micrometers to millimeters. As another example, thepores of the matrix material can have a diameter of about 100 μm toabout 600 μm. As another example, the pores of the matrix material canhave a diameter of about 150 μm, about 200 μm, about 250 μm, about 300μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, or about 550μm. It is understood that the pores of a scaffold material can have thesame, approximately the same, or different average diameters betweendifferent components or portions of a scaffold.

A scaffold, or portion or component thereof, can be produced fromproteins (e.g. extracellular matrix proteins such as fibrin, collagen,and fibronectin), polymers (e.g., polyvinylpyrrolidone), polysaccharides(e.g. alginate), hyaluronic acid, or analogs, mixtures, combinations,and derivatives of the above.

For example, a scaffold, or portion or component thereof, can be formedof synthetic polymers. Such synthetic polymers include, but are notlimited to, poly(ethylene) glycol, bioerodible polymers (e.g.,poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide),poly(caprolactone), polyester (e.g., poly-(L-lactic acid),polyanhydride, polyglactin, polyglycolic acid), polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates), polyphosphazene, degradablepolyurethanes, non-erodible polymers (e.g., polyacrylates,ethylene-vinyl acetate polymers and other acyl substituted celluloseacetates and derivatives thereof), non-erodible polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinylpyrrolidone, poly(vinylimidazole), chlorosulphonated polyolifins,polyethylene oxide, polyvinyl alcohol (e.g., polyvinyl alcohol sponge),synthetic marine adhesive proteins, Teflon®, nylon, or analogs,mixtures, combinations (e.g., polyethylene oxide-polypropylene glycolblock copolymer; poly(D,L-lactide-co-glycolide) fiber matrix), andderivatives of the above.

As another example, suitable scaffold materials include, for example, acollagen gel, polyvinyl alcohol, a marine adhesive protein, a PLG fibermatrix, a polyglactin fiber, a calcium alginate gel, a polyglycolicacid, polyester (e.g., poly-(L-lactic acid) or a polyanhydride), apolysaccharide (e.g. alginate), chitosan, polyphosphazene, polyacrylate,polyethylene oxide-polypropylene glycol block copolymer, fibrin,collagen, and fibronectin, polyvinylpyrrolidone, hyaluronic acid,poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide),poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyamino acids, polyortho esters, polyacetals, polycyanoacrylates),polyurethanes, polyacrylates, ethylene-vinyl acetate polymers and otheracyl substituted cellulose acetates and derivatives thereof),polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinylimidazole), chlorosulphonated polyolifins, polyethylene oxide,polyvinyl alcohol, Teflon®, nylon, and analogs, mixtures, combinationsand derivatives of any of the above.

As another example, a scaffold, or portion or component thereof, can beformed of naturally occurring polymers or natively derived polymers.Such polymers include, but are not limited to, agarose, alginate (e.g.,calcium alginate gel), fibrin, fibrinogen, fibronectin, collagen (e.g.,a collagen gel), gelatin, hyaluronic acid, chitin, and other suitablepolymers and biopolymers, or analogs, mixtures, combinations, andderivatives of the above. Also, a scaffold, or portion or componentthereof, can be formed from a mixture of naturally occurring biopolymersand synthetic polymers.

As another example, a scaffold can include polycarboxylates,polyanhydrides, poly(α-hydroxyesters), poly(ethylene terephthalates),poly(carbonates), poly(amides), poly(lactones), a poly(saccharides) andpoly(acrylates).

A scaffold, or portion or component thereof, can comprise a crystallineor mineral component. For example, a scaffold, or portion or componentthereof, can include the inorganic mineral hydroxyapatite (also known ashydroxylapatite). About seventy percent of natural bone is made up ofhydroxyapatite. In some embodiments, a scaffold, or portion or componentthereof, comprises a ground natural substance containing hydroxyapatite,such as bone. In some embodiments, a scaffold, or portion or componentthereof, comprises substantially pure hydroxyapatite.

A scaffold, or portion or component thereof, can comprise a compositematerial comprising at least two components described above. As anexample, a composite scaffold material can comprise at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten, or more, components. The plurality ofcomponents can be homogenously mixed throughout the scaffold,heterologously mixed throughout the scaffold, or separated intodifferent layers of the scaffold, or a combination thereof.

In some embodiments, a scaffold, or portion or component thereof,comprises polycaprolactone, polylactide, polyglycolide,poly(lactide-glycolide), poly(propylene fumarate), poly(caprolactonefumarate), polyethylene glycol, poly(glycolide-co-caprolactone), ormixtures thereof.

For example, a scaffold, or portion or component thereof, can be formedin whole or in part of polycaprolactone or a mixture, composite, orderivative thereof. Polycaprolactone can be a particularly usefulmaterial where the scaffolds can be prepared by the methods described inU.S. Pat Pub No. 2003/0069718, U.S. Pat Pub No. 2006/0276925, U.S. PatPub No. 2008/0195211, U.S. Pat Pub No. 2008/0215093, or U.S. patentapplication Ser. No. 13/036,470, all are incorporated herein byreference in their entireties.

A scaffold, as described herein, can be any material suitable formineral coating. For example, a scaffold can include a bead, amicrosphere, a cage, or a modular scaffold. The mineral coated scaffoldscan be prepared by the methods described in U.S. patent Ser. Nos.13/407,441; 13/879,178; and 13/036,470 and are incorporated herein byreference.

Modified Simulated Body Fluid

A modified simulated body fluid as described herein can be a solutionincluding ionic constituents of blood plasma. In some embodiments, themodified simulated body fluid does not comprise organic components.Inorganic minerals suitable for producing a calcium-containing mineralcoating include various bone mineral ions, such as, but not limited tocalcium and phosphate and combinations of bone mineral ions, such ascalcium-phosphates. A modified simulated body fluid can be as describedin U.S. application Ser. Nos. 13/407,441; 13/879,178; and 13/036,470 andare incorporated by reference.

A modified simulated body fluid as described herein can be used tomineral coat a scaffold. For example, the scaffold can be immersed andincubated in a modified simulated body fluid. As another example, themodified simulated body fluid can be replaced, replenished, or removedand added at least about once a day; at least about twice per day; or atleast about three times per day. As another example, the modifiedsimulated body fluid can be replaced at least about once every day; atleast about once every two days; at least once every three days; atleast once every four days; at least once every five days; at least onceevery six days; or at least once every seven days.

As described herein, a modified simulated body fluid can be a solutionof ionic constituents of blood plasma, with double the concentrations ofcalcium and phosphate ions.

As described herein, a modified simulated body fluid can be a solutioncomprising NaCl, KCl, MgCl₂, MgSO₄, NaHCO₃, CaCl₂, and KH₂PO₄.

A modified simulated body fluid can include at least about 1 mM NaCl.For example, a modified simulated body fluid can include at least about1 mM NaCl; at least about 10 mM NaCl; at least about 20 mM NaCl; atleast about 30 mM NaCl; at least about 40 mM NaCl; at least about 50 mMNaCl; at least about 60 mM NaCl; at least about 70 mM NaCl; at leastabout 80 mM NaCl; at least about 90 mM NaCl; at least about 100 mM NaCl;at least about 110 mM NaCl; at least about 120 mM NaCl; at least about130 mM NaCl; at least about 140 mM NaCl; at least about 150 mM NaCl; atleast about 160 mM NaCl; at least about 170 mM NaCl; at least about 180mM NaCl; at least about 190 mM NaCl; at least about 200 mM NaCl; atleast about 300 mM NaCl; at least about 400 mM NaCl; at least about 500mM NaCl; at least about 600 mM NaCl; at least about 700 mM NaCl; atleast about 800 mM NaCl; at least about 900 mM NaCl; at least about 1000mM NaCl; at least about 1100 mM NaCl; at least about 1200 mM NaCl; atleast about 1300 mM NaCl; at least about 1400 mM NaCl; at least about1500 mM NaCl; at least about 1600 mM NaCl; at least about 1700 mM NaCl;at least about 1800 mM NaCl; at least about 1900 mM NaCl; or at leastabout 2000 mM NaCl. It is understood that recitation of the abovediscrete values includes a range between each recited value.

As another example, a modified simulated body fluid can include about 1mM NaCl, about 10 mM NaCl; about 20 mM NaCl; about 30 mM NaCl; about 40mM NaCl; about 50 mM NaCl; about 60 mM NaCl; about 70 mM NaCl; about 80mM NaCl; about 90 mM NaCl; about 100 mM NaCl; about 110 mM NaCl; about120 mM NaCl; about 130 mM NaCl; about 140 mM NaCl; about 150 mM NaCl;about 160 mM NaCl; about 170 mM NaCl; about 180 mM NaCl; about 190 mMNaCl; about 200 mM NaCl; about 300 mM NaCl; about 400 mM NaCl; about 500mM NaCl; about 600 mM NaCl; about 700 mM NaCl; about 800 mM NaCl; about900 mM NaCl; about 1000 mM NaCl; about 1100 mM NaCl; about 1200 mM NaCl;about 1300 mM NaCl; about 1400 mM NaCl; about 1500 mM NaCl; about 1600mM NaCl; about 1700 mM NaCl; about 1800 mM NaCl; about 1900 mM NaCl; orabout 2000 mM NaCl. It is understood that recitation of the abovediscrete values includes a range between each recited value.

A modified simulated body fluid can include at least about 0.4 mM KCl.For example, a modified simulated body fluid can include at least about0.4 mM KCl; at least about 1 mM KCl; at least about 2 mM KCl; at leastabout 3 mM KCl; at least about 4 mM KCl; at least about 5 mM KCl; atleast about 6 mM KCl; at least about 7 mM KCl; at least about 8 mM KCl;at least about 9 mM KCl; at least about 10 mM KCl; at least about 11 mMKCl; at least about 12 mM KCl; at least about 13 mM KCl; at least about14 mM KCl; at least about 15 mM KCl; at least about 16 mM KCl; at leastabout 17 mM KCl; at least about 18 mM KCl; at least about 19 mM KCl; atleast about 20 mM KCl; at least about 21 mM KCl; at least about 22 mMKCl; at least about 23 mM KCl; at least about 24 mM KCl; at least about25 mM KCl; at least about 26 mM KCl; at least about 27 mM KCl; at leastabout 28 mM KCl; at least about 29 mM KCl; at least about 30 mM KCl; atleast about 31 mM KCl; at least about 32 mM KCl; at least about 33 mMKCl; at least about 34 mM KCl; at least about 35 mM KCl; at least about36 mM KCl; at least about 37 mM KCl; at least about 38 mM KCl; at leastabout 39 mM KCl; at least about 40 mM KCl; at least about 41 mM KCl; atleast about 42 mM KCl; at least about 43 mM KCl; at least about 44 mMKCl; at least about 45 mM KCl; at least about 46 mM KCl; at least about47 mM KCl; at least about 48 mM KCl; at least about 49 mM KCl; at leastabout 50 mM KCl; at least about 51 mM KCl; at least about 52 mM KCl; atleast about 53 mM KCl; at least about 54 mM KCl; at least about 55 mMKCl; at least about 56 mM KCl; at least about 57 mM KCl; at least about58 mM KCl; at least about 59 mM KCl; at least about 60 mM KCl; at leastabout 61 mM KCl; at least about 62 mM KCl; at least about 63 mM KCl; atleast about 64 mM KCl; at least about 65 mM KCl; at least about 66 mMKCl; at least about 67 mM KCl; at least about 68 mM KCl; at least about69 mM KCl; at least about 70 mM KCl; at least about 71 mM KCl; at leastabout 72 mM KCl; at least about 73 mM KCl; at least about 74 mM KCl; atleast about 75 mM KCl; at least about 76 mM KCl; at least about 77 mMKCl; at least about 78 mM KCl; at least about 79 mM KCl; or at leastabout 80 mM KCl. It is understood that recitation of the above discretevalues includes a range between each recited value.

As another example, a modified simulated body fluid can include about0.4 mM KCl; about 1 mM KCl; about 2 mM KCl; about 3 mM KCl; about 4 mMKCl; about 5 mM KCl; about 6 mM KCl; about 7 mM KCl; about 8 mM KCl;about 9 mM KCl; about 10 mM KCl; about 11 mM KCl; about 12 mM KCl; about13 mM KCl; about 14 mM KCl; about 15 mM KCl; about 16 mM KCl; about 17mM KCl; about 18 mM KCl; about 19 mM KCl; about 20 mM KCl; about 21 mMKCl; about 22 mM KCl; about 23 mM KCl; about 24 mM KCl; about 25 mM KCl;about 26 mM KCl; about 27 mM KCl; about 28 mM KCl; about 29 mM KCl;about 30 mM KCl; about 31 mM KCl; about 32 mM KCl; about 33 mM KCl;about 34 mM KCl; about 35 mM KCl; about 36 mM KCl; about 37 mM KCl;about 38 mM KCl; about 39 mM KCl; about 40 mM; about 41 mM KCl; about 42mM KCl; about 43 mM KCl; about 44 mM KCl; about 45 mM KCl; about 46 mMKCl; about 47 mM KCl; about 48 mM KCl; about 49 mM KCl; about 50 mM KCl;about 51 mM KCl; about 52 mM KCl; about 53 mM KCl; about 54 mM KCl;about 55 mM KCl; about 56 mM KCl; about 57 mM KCl; about 58 mM KCl;about 59 mM KCl; about 60 mM KCl; about 61 mM KCl; about 62 mM KCl;about 63 mM KCl; about 64 mM KCl; about 65 mM KCl; about 66 mM KCl;about 67 mM KCl; about 68 mM KCl; about 69 mM KCl; about 70 mM KCl;about 71 mM KCl; about 72 mM KCl; about 73 mM KCl; about 74 mM KCl;about 75 mM KCl; about 76 mM KCl; about 77 mM KCl; about 78 mM KCl;about 79 mM KCl; or about 80 mM KCl. It is understood that recitation ofthe above discrete values includes a range between each recited value.

A modified simulated body fluid can include at least about 0.1 mM MgCl₂.For example, a modified simulated body fluid can include at least about0.1 mM MgCl₂; at least about 0.25 mM MgCl₂; at least about 0.5 mM MgCl₂;at least about 1 mM MgCl₂; at least about 1.25 mM MgCl₂; at least about1.5 mM MgCl₂; at least about 1.75 mM MgCl₂; at least about 2 mM MgCl₂;at least about 2.25 mM MgCl₂; at least about 2.5 mM MgCl₂; at leastabout 2.75 mM MgCl₂; at least about 3 mM MgCl₂; at least about 3.25 mMMgCl₂; at least about 3.5 mM MgCl₂; at least about 3.75 mM MgCl₂; atleast about 4 mM MgCl₂; at least about 4.25 mM MgCl₂; at least about 4.5mM MgCl₂; at least about 4.75 mM MgCl₂; at least about 5 mM MgCl₂; atleast about 5.25 mM MgCl₂; at least about 5.5 mM MgCl₂; at least about5.75 mM MgCl₂; at least about 6 mM MgCl₂; at least about 6.25 mM MgCl₂;at least about 6.5 mM MgCl₂; at least about 6.75 mM MgCl₂; at leastabout 7 mM MgCl₂; at least about 7.25 mM MgCl₂; at least about 7.5 mMMgCl₂; at least about 7.75 mM MgCl₂; at least about 8 mM MgCl₂; at leastabout 8.25 mM MgCl₂; at least about 8.5 mM MgCl₂; at least about 8.75 mMMgCl₂; at least about 9 mM MgCl₂; at least about 9.25 mM MgCl₂; at leastabout 9.5 mM MgCl₂; at least about 9.75 mM MgCl₂; at least about 10 mMMgCl₂; at least about 11 mM MgCl₂; at least about 12 mM MgCl₂; at leastabout 13 mM MgCl₂; at least about 14 mM MgCl₂; at least about 15 mMMgCl₂; at least about 16 mM MgCl₂; at least about 17 mM MgCl₂; at leastabout 18 mM MgCl₂; at least about 19 mM MgCl₂; at least about 20 mMMgCl₂; at least about 21 mM MgCl₂; at least about 22 mM MgCl₂; at leastabout 23 mM MgCl₂; at least about 24 mM MgCl₂; at least about 25 mMMgCl₂; at least about 26 mM MgCl₂; at least about 27 mM MgCl₂; at leastabout 28 mM MgCl₂; at least about 29 mM MgCl₂; at least about 30 mMMgCl₂; at least about 31 mM MgCl₂; at least about 32 mM MgCl₂; at leastabout 33 mM MgCl₂; at least about 34 mM MgCl₂; at least about 35 mMMgCl₂; at least about 36 mM MgCl₂; at least about 37 mM MgCl₂; at leastabout 38 mM MgCl₂; at least about 39 mM MgCl₂; at least about 40 mMMgCl₂; at least about 41 mM MgCl₂; at least about 42 mM MgCl₂; at leastabout 43 mM MgCl₂; at least about 44 mM MgCl₂; at least about 45 mMMgCl₂; at least about 46 mM MgCl₂; at least about 47 mM MgCl₂; at leastabout 48 mM MgCl₂; at least about 49 mM MgCl₂; or at least about 50 mMMgCl₂. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

As another example, a modified simulated body fluid can include about0.1 mM MgCl₂; at least about 0.25 mM MgCl₂; about 0.5 mM MgCl₂; about 1mM MgCl₂; about 1.25 mM MgCl₂; about 1.5 mM MgCl₂; about 1.75 mM MgCl₂;about 2 mM MgCl₂; about 2.25 mM MgCl₂; about 2.5 mM MgCl₂; about 2.75 mMMgCl₂; about 3 mM MgCl₂; about 3.25 mM MgCl₂; about 3.5 mM MgCl₂; about3.75 mM MgCl₂; about 4 mM MgCl₂; about 4.25 mM MgCl₂; about 4.5 mMMgCl₂; about 4.75 mM MgCl₂; about 5 mM MgCl₂; about 5.25 mM MgCl₂; about5.5 mM MgCl₂; about 5.75 mM MgCl₂; about 6 mM MgCl₂; about 6.25 mMMgCl₂; about 6.5 mM MgCl₂; about 6.75 mM MgCl₂; about 7 mM MgCl₂; about7.25 mM MgCl₂; about 7.5 mM MgCl₂; about 7.75 mM MgCl₂; about 8 mMMgCl₂; about 8.25 mM MgCl₂; about 8.5 mM MgCl₂; about 8.75 mM MgCl₂;about 9 mM MgCl₂; about 9.25 mM MgCl₂; about 9.5 mM MgCl₂; about 9.75 mMMgCl₂; about 10 mM MgCl₂; about 11 mM MgCl₂; about 12 mM MgCl₂; about 13mM MgCl₂; about 14 mM MgCl₂; about 15 mM MgCl₂; about 16 mM MgCl₂;

-   -   about 17 mM MgCl₂; about 18 mM MgCl₂; about 19 mM MgCl₂; about        20 mM MgCl₂; about 21 mM MgCl₂; about 22 mM MgCl₂; about 23 mM        MgCl₂; about 24 mM MgCl₂; about 25 mM MgCl₂; about 26 mM MgCl₂;        about 27 mM MgCl₂;    -   about 28 mM MgCl₂; about 29 mM MgCl₂; about 30 mM MgCl₂; about        31 mM MgCl₂; about 32 mM MgCl₂; about 33 mM MgCl₂; about 34 mM        MgCl₂; about 35 mM MgCl₂; about 36 mM MgCl₂; about 37 mM MgCl₂;        about 38 mM MgCl₂;    -   about 39 mM MgCl₂; about 40 mM MgCl₂; about 41 mM MgCl₂; about        42 mM MgCl₂; about 43 mM MgCl₂; about 44 mM MgCl₂; about 45 mM        MgCl₂; about 46 mM MgCl₂; about 47 mM MgCl₂; about 48 mM MgCl₂;        about 49 mM MgCl₂; or about 50 mM MgCl₂. It is understood that        recitation of the above discrete values includes a range between        each recited value.

A modified simulated body fluid can include at least about 0.05 mMMgSO₄. For example, a modified simulated body fluid can include at leastabout 0.05 mM MgSO₄; at least about 0.25 mM MgSO₄; at least about 0.5 mMMgSO₄; at least about 0.75 mM MgSO₄; at least about 1 mM MgSO₄; at leastabout 1.25 mM MgSO₄; at least about 1.5 mM MgSO₄; at least about 1.75 mMMgSO₄; at least about 2 mM MgSO₄; at least about 2.25 mM MgSO₄; at leastabout 2.5 mM MgSO₄; at least about 2.75 mM MgSO₄; at least about 3 mMMgSO₄; at least about 3.25 mM MgSO₄; at least about 3.5 mM MgSO₄; atleast about 4 mM MgSO₄; at least about 4.25 mM MgSO₄; at least about 4.5mM MgSO₄; at least about 4.75 mM MgSO₄; at least about 5 mM MgSO₄; atleast about 6 mM MgSO₄; at least about 7 mM MgSO₄; at least about 8 mMMgSO₄; at least about 9 mM MgSO₄; at least about 10 mM MgSO₄; at leastabout 11 mM MgSO₄; at least about 12 mM MgSO₄; at least about 13 mMMgSO₄; at least about 14 mM MgSO₄; at least about 15 mM MgSO₄; at leastabout 16 mM MgSO₄; at least about 17 mM MgSO₄; at least about 18 mMMgSO₄; at least about 19 mM MgSO₄; at least about 20 mM MgSO₄; at leastabout 21 mM MgSO₄; at least about 22 mM MgSO₄; at least about 23 mMMgSO₄; at least about 24 mM MgSO₄; at least about 25 mM MgSO₄; at leastabout 26 mM MgSO₄; at least about 27 mM MgSO₄; at least about 28 mMMgSO₄; at least about 29 mM MgSO₄; at least about 30 mM MgSO₄; at leastabout 31 mM MgSO₄; at least about 32 mM MgSO₄; at least about 33 mMMgSO₄; at least about 34 mM MgSO₄; at least about 35 mM MgSO₄; at leastabout 36 mM MgSO₄; at least about 37 mM MgSO₄; at least about 38 mMMgSO₄; at least about 39 mM MgSO₄; at least about 40 mM MgSO₄; at leastabout 41 mM MgSO₄; at least about 42 mM MgSO₄; at least about 43 mMMgSO₄; at least about 44 mM MgSO₄; at least about 45 mM MgSO₄; at leastabout 46 mM MgSO₄; at least about 47 mM MgSO₄; at least about 48 mMMgSO₄; at least about 49 mM MgSO₄; or at least about 50 mM MgSO₄. It isunderstood that recitation of the above discrete values includes a rangebetween each recited value.

As another example, a modified simulated body fluid can include about0.05 mM MgSO₄; about 0.25 mM MgSO₄; about 0.5 mM MgSO₄; about 0.75 mMMgSO₄; about 1 mM MgSO₄; about 1.25 mM MgSO₄; about 1.5 mM MgSO₄;

-   -   about 1.75 mM MgSO₄; about 2 mM MgSO₄; about 2.25 mM MgSO₄;        about 2.5 mM MgSO₄; about 2.75 mM MgSO₄; about 3 mM MgSO₄; about        3.25 mM MgSO₄; about 3.5 mM MgSO₄; about 4 mM MgSO₄; about 4.25        mM MgSO₄;    -   about 4.5 mM MgSO₄; about 4.75 mM MgSO₄; about 5 mM MgSO₄; about        6 mM MgSO₄; about 7 mM MgSO₄; about 8 mM MgSO₄; about 9 mM        MgSO₄; about 10 mM MgSO₄; about 11 mM MgSO₄; about 12 mM MgSO₄;        about 13 mM MgSO₄;    -   about 14 mM MgSO₄; about 15 mM MgSO₄; about 16 mM MgSO₄; about        17 mM MgSO₄; about 18 mM MgSO₄; about 19 mM MgSO₄; about 20 mM        MgSO₄;    -   about 21 mM MgSO₄; about 22 mM MgSO₄; about 23 mM MgSO₄; about        24 mM MgSO₄; about 25 mM MgSO₄; about 26 mM MgSO₄; about 27 mM        MgSO₄;    -   about 28 mM MgSO₄; about 29 mM MgSO₄; about 30 mM MgSO₄; about        31 mM MgSO₄; about 32 mM MgSO₄; about 33 mM MgSO₄; about 34 mM        MgSO₄;    -   about 35 mM MgSO₄; about 36 mM MgSO₄; about 37 mM MgSO₄; about        38 mM MgSO₄; about 39 mM MgSO₄; about 40 mM MgSO₄; about 41 mM        MgSO₄;    -   about 42 mM MgSO₄; about 43 mM MgSO₄; about 44 mM MgSO₄; about        45 mM MgSO₄; about 46 mM MgSO₄; about 47 mM MgSO₄; about 48 mM        MgSO₄;    -   about 49 mM MgSO₄; or about 50 mM MgSO₄. It is understood that        recitation of the above discrete values includes a range between        each recited value.

A modified simulated body fluid can include at least about 0.4 mMNaHCO₃. For example, a modified simulated body fluid can include atleast about 0.4 mM NaHCO₃; at least about 0.6 mM NaHCO₃; at least about0.8 mM NaHCO₃; at least about 1.0 mM NaHCO₃; at least about 1.2 mMNaHCO₃; at least about 1.4 mM NaHCO₃; at least about 1.6 mM NaHCO₃; atleast about 1.8 mM NaHCO₃; at least about 2.0 mM NaHCO₃; at least about2.2 mM NaHCO₃; at least about 2.4 mM NaHCO₃; at least about 2.6 mMNaHCO₃; at least about 2.8 mM NaHCO₃; at least about 3.0 mM NaHCO₃; atleast about 3.2 mM NaHCO₃; at least about 3.4 mM NaHCO₃; at least about3.6 mM NaHCO₃; at least about 3.8 mM NaHCO₃; at least about 4.0 mMNaHCO₃; at least about 4.2 mM NaHCO₃; at least about 4.4 mM NaHCO₃; atleast about 4.6 mM NaHCO₃; at least about 4.8 mM NaHCO₃; at least about5.0 mM NaHCO₃; at least about 5.2 mM NaHCO₃; at least about 5.4 mMNaHCO₃; at least about 5.6 mM NaHCO₃; at least about 5.8 mM NaHCO₃; atleast about 6.0 mM NaHCO₃; at least about 6.2 mM NaHCO₃; at least about6.4 mM NaHCO₃; at least about 6.6 mM NaHCO₃; at least about 6.8 mMNaHCO₃; at least about 7.0 mM NaHCO₃; at least about 7.2 mM NaHCO₃; atleast about 7.4 mM NaHCO₃; at least about 7.6 mM NaHCO₃; at least about7.8 mM NaHCO₃; at least about 8.0 mM NaHCO₃; at least about 8.2 mMNaHCO₃; at least about 8.4 mM NaHCO₃; at least about 8.6 mM NaHCO₃; atleast about 8.8 mM NaHCO₃; at least about 9.0 mM NaHCO₃; at least about10 mM NaHCO₃; at least about 20 mM NaHCO₃; at least about 30 mM NaHCO₃;at least about 40 mM NaHCO₃; at least about 50 mM NaHCO₃; at least about60 mM NaHCO₃; at least about 70 mM NaHCO₃; at least about 80 mM NaHCO₃;at least about 90 mM NaHCO₃; at least about 100 mM NaHCO₃; at leastabout 200 mM NaHCO₃; at least about 300 mM NaHCO₃; at least about 400 mMNaHCO₃; at least about 500 mM NaHCO₃; at least about 600 mM NaHCO₃; atleast about 700 mM NaHCO₃; at least about 800 mM NaHCO₃; at least about900 mM NaHCO₃; or at least about 1000 mM NaHCO₃.

As another example, a modified simulated body fluid can include about0.4 mM NaHCO₃; about 0.6 mM NaHCO₃; about 0.8 mM NaHCO₃; about 1.0 mMNaHCO₃; about 1.2 mM NaHCO₃; about 1.4 mM NaHCO₃; about 1.6 mM NaHCO₃;about 1.8 mM NaHCO₃; about 2.0 mM NaHCO₃; about 2.2 mM NaHCO₃; about 2.4mM NaHCO₃; about 2.6 mM NaHCO₃; about 2.8 mM NaHCO₃; about 3.0 mMNaHCO₃; about 3.2 mM NaHCO₃; about 3.4 mM NaHCO₃; about 3.6 mM NaHCO₃;about 3.8 mM NaHCO₃; about 4.0 mM NaHCO₃; about 4.2 mM NaHCO₃; about 4.4mM NaHCO₃; about 4.6 mM NaHCO₃; about 4.8 mM NaHCO₃; about 5.0 mMNaHCO₃; about 5.2 mM NaHCO₃; about 5.4 mM NaHCO₃; about 5.6 mM NaHCO₃;about 5.8 mM NaHCO₃; about 6.0 mM NaHCO₃; about 6.2 mM NaHCO₃; about 6.4mM NaHCO₃; about 6.6 mM NaHCO₃; about 6.8 mM NaHCO₃; about 7.0 mMNaHCO₃; about 7.2 mM NaHCO₃; about 7.4 mM NaHCO₃; about 7.6 mM NaHCO₃;about 7.8 mM NaHCO₃; about 8.0 mM NaHCO₃; about 8.2 mM NaHCO₃; about 8.4mM NaHCO₃; about 8.6 mM NaHCO₃; about 8.8 mM NaHCO₃; about 9.0 mMNaHCO₃; about 10 mM NaHCO₃; about 20 mM NaHCO₃; about 30 mM NaHCO₃;about 40 mM NaHCO₃; about 50 mM NaHCO₃; about 60 mM NaHCO₃; about 70 mMNaHCO₃; about 80 mM NaHCO₃; about 90 mM NaHCO₃; about 100 mM NaHCO₃;about 200 mM NaHCO₃; about 300 mM NaHCO₃; about 400 mM NaHCO₃; about 500mM NaHCO₃; about 600 mM NaHCO₃; about 700 mM NaHCO₃; about 800 mMNaHCO₃; about 900 mM NaHCO₃; or about 1000 mM NaHCO₃. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

A modified simulated body fluid can include at least about 0.5 mM CaCl₂.For example, a modified simulated body fluid can include at least about0.5 mM CaCl₂; at least about 1.0 mM CaCl₂; at least about 1.5 mM CaCl₂;at least about 2.0 mM CaCl₂; at least about 2.5 mM CaCl₂; at least about3.0 mM CaCl₂; at least about 3.5 mM CaCl₂; at least about 4.0 mM CaCl₂;at least about 4.5 mM CaCl₂; at least about 5.0 mM CaCl₂; at least about5.5 mM CaCl₂; at least about 6.0 mM CaCl₂; at least about 6.5 mM CaCl₂;at least about 7.0 mM CaCl₂; at least about 7.5 mM CaCl₂; at least about8.0 mM CaCl₂; at least about 8.5 mM CaCl₂; at least about 9.0 mM CaCl₂;at least about 9.5 mM CaCl₂; at least about 10.0 mM CaCl₂; at leastabout 10.5 mM CaCl₂; at least about 11.0 mM CaCl₂; at least about 11.5mM CaCl₂; at least about 12.0 mM CaCl₂; at least about 12.5 mM CaCl₂; atleast about 13.0 mM CaCl₂; at least about 13.5 mM CaCl₂; at least about14.0 mM CaCl₂; at least about 14.5 mM CaCl₂; at least about 15.0 mMCaCl₂; at least about 15.5 mM CaCl₂; at least about 16.0 mM CaCl₂; atleast about 16.5 mM CaCl₂; at least about 17.0 mM CaCl₂; at least about17.5 mM CaCl₂; at least about 18.0 mM CaCl₂; at least about 18.5 mMCaCl₂; at least about 19.0 mM CaCl₂; at least about 19.5 mM CaCl₂; atleast about 20.0 mM CaCl₂; at least about 25 mM CaCl₂; at least about 30mM CaCl₂; at least about 35 mM CaCl₂; at least about 40 mM CaCl₂; atleast about 45 mM CaCl₂; or at least about 50 mM CaCl₂. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

As another example, a modified simulated body fluid can include about0.5 mM CaCl₂; about 1.0 mM CaCl₂; about 1.5 mM CaCl₂; about 2.0 mMCaCl₂; about 2.5 mM CaCl₂; about 3.0 mM CaCl₂; about 3.5 mM CaCl₂; about4.0 mM CaCl₂; about 4.5 mM CaCl₂; about 5.0 mM CaCl₂; about 5.5 mMCaCl₂; about 6.0 mM CaCl₂; about 6.5 mM CaCl₂; about 7.0 mM CaCl₂; about7.5 mM CaCl₂; about 8.0 mM CaCl₂; about 8.5 mM CaCl₂; about 9.0 mMCaCl₂; about 9.5 mM CaCl₂; about 10.0 mM CaCl₂; about 10.5 mM CaCl₂;about 11.0 mM CaCl₂; about 11.5 mM CaCl₂; about 12.0 mM CaCl₂; about12.5 mM CaCl₂; about 13.0 mM CaCl₂; about 13.5 mM CaCl₂; about 14.0 mMCaCl₂; about 14.5 mM CaCl₂; about 15.0 mM CaCl₂; about 15.5 mM CaCl₂;about 16.0 mM CaCl₂; about 16.5 mM CaCl₂; about 17.0 mM CaCl₂; about17.5 mM CaCl₂; about 18.0 mM CaCl₂; about 18.5 mM CaCl₂; about 19.0 mMCaCl₂; about 19.5 mM CaCl₂; about 20.0 mM CaCl₂; about 25 mM CaCl₂;about 30 mM CaCl₂; about 35 mM CaCl₂; about 40 mM CaCl₂; about 45 mMCaCl₂; or about 50 mM CaCl₂. It is understood that recitation of theabove discrete values includes a range between each recited value.

A modified simulated body fluid can include at least about 0.2 mMKH₂PO₄. For example, a modified simulated body fluid can include atleast about 0.2 mM KH₂PO₄; at least about 0.4 mM KH₂PO₄; at least about0.6 mM KH₂PO₄; at least about 0.8 mM KH₂PO₄; at least about 1.0 mMKH₂PO₄; at least about 1.2 mM KH₂PO₄; at least about 1.4 mM KH₂PO₄; atleast about 1.6 mM KH₂PO₄; at least about 1.8 mM KH₂PO₄; at least about2.0 mM KH₂PO₄; at least about 2.2 mM KH₂PO₄; at least about 2.4 mMKH₂PO₄; at least about 2.6 mM KH₂PO₄; at least about 2.8 mM KH₂PO₄; atleast about 3.0 mM KH₂PO₄; at least about 3.2 mM KH₂PO₄; at least about3.4 mM KH₂PO₄; at least about 3.6 mM KH₂PO₄; at least about 3.8 mMKH₂PO₄; at least about 4.0 mM KH₂PO₄; at least about 4.2 mM KH₂PO₄; atleast about 4.4 mM KH₂PO₄; at least about 4.6 mM KH₂PO₄; at least about4.8 mM KH₂PO₄; at least about 5.0 mM KH₂PO₄; at least about 5.2 mMKH₂PO₄; at least about 5.4 mM KH₂PO₄; at least about 5.6 mM KH₂PO₄; atleast about 5.8 mM KH₂PO₄; at least about 6.0 mM KH₂PO₄; at least about6.2 mM KH₂PO₄; at least about 6.4 mM KH₂PO₄; at least about 6.8 mMKH₂PO₄; at least about 7.0 mM KH₂PO₄; at least about 7.2 mM KH₂PO₄; atleast about 7.4 mM KH₂PO₄; at least about 7.6 mM KH₂PO₄; at least about7.8 mM KH₂PO₄; at least about 8.0 mM KH₂PO₄; at least about 8.2 mMKH₂PO₄; at least about 8.4 mM KH₂PO₄; at least about 8.6 mM KH₂PO₄; atleast about 8.8 mM KH₂PO₄; at least about 9.0 mM KH₂PO₄; at least about9.2 mM KH₂PO₄; at least about 9.4 mM KH₂PO₄; at least about 9.6 mMKH₂PO₄; at least about 9.8 mM KH₂PO₄; at least about 10.0 mM KH₂PO₄; atleast about 20 mM KH₂PO₄; at least about 30 mM KH₂PO₄; at least about 40mM KH₂PO₄; at least about 50 mM KH₂PO₄; at least about 60 mM KH₂PO₄; atleast about 70 mM KH₂PO₄; at least about 80 mM KH₂PO₄; at least about 90mM KH₂PO₄; at least about 100 mM KH₂PO₄; at least about 110 mM KH₂PO₄;at least about 120 mM KH₂PO₄; at least about 130 mM KH₂PO₄; at leastabout 140 mM KH₂PO₄; at least about 150 mM KH₂PO₄; at least about 160 mMKH₂PO₄; at least about 170 mM KH₂PO₄; at least about 180 mM KH₂PO₄; atleast about 190 mM KH₂PO₄; or at least about 200 mM KH₂PO₄. It isunderstood that recitation of the above discrete values includes a rangebetween each recited value.

As another example, a modified simulated body fluid can include about0.2 mM KH₂PO₄; about 0.4 mM KH₂PO₄; about 0.6 mM KH₂PO₄; about 0.8 mMKH₂PO₄; about 1.0 mM KH₂PO₄; about 1.2 mM KH₂PO₄; about 1.4 mM KH₂PO₄;about 1.6 mM KH₂PO₄; about 1.8 mM KH₂PO₄; about 2.0 mM KH₂PO₄; about 2.2mM KH₂PO₄; about 2.4 mM KH₂PO₄; about 2.6 mM KH₂PO₄; about 2.8 mMKH₂PO₄; about 3.0 mM KH₂PO₄; about 3.2 mM KH₂PO₄; about 3.4 mM KH₂PO₄;about 3.6 mM KH₂PO₄; about 3.8 mM KH₂PO₄; about 4.0 mM KH₂PO₄; about 4.2mM KH₂PO₄; about 4.4 mM KH₂PO₄; about 4.6 mM KH₂PO₄; about 4.8 mMKH₂PO₄; about 5.0 mM KH₂PO₄; about 5.2 mM KH₂PO₄; about 5.4 mM KH₂PO₄;about 5.6 mM KH₂PO₄; about 5.8 mM KH₂PO₄; about 6.0 mM KH₂PO₄; about 6.2mM KH₂PO₄; about 6.4 mM KH₂PO₄; about 6.8 mM KH₂PO₄; about 7.0 mMKH₂PO₄; about 7.2 mM KH₂PO₄; about 7.4 mM KH₂PO₄; about 7.6 mM KH₂PO₄;about 7.8 mM KH₂PO₄; about 8.0 mM KH₂PO₄; about 8.2 mM KH₂PO₄; about 8.4mM KH₂PO₄; about 8.6 mM KH₂PO₄; about 8.8 mM KH₂PO₄; about 9.0 mMKH₂PO₄; about 9.2 mM KH₂PO₄; about 9.4 mM KH₂PO₄; about 9.6 mM KH₂PO₄;about 9.8 mM KH₂PO₄; about 10.0 mM KH₂PO₄; about 20 mM KH₂PO₄; about 30mM KH₂PO₄; about 40 mM KH₂PO₄; about 50 mM KH₂PO₄; about 60 mM KH₂PO₄;about 70 mM KH₂PO₄; about 80 mM KH₂PO₄; about 90 mM KH₂PO₄; about 100 mMKH₂PO₄; about 110 mM KH₂PO₄; about 120 mM KH₂PO₄; about 130 mM KH₂PO₄;about 140 mM KH₂PO₄; about 150 mM KH₂PO₄; about 160 mM KH₂PO₄; about 170mM KH₂PO₄; about 180 mM KH₂PO₄; about 190 mM KH₂PO₄; or about 200 mMKH₂PO₄. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

In some embodiments, the solution can comprise a surfactant, which canchange the morphology of the calcium-containing mineral layer. Anysurfactant now known or later discovered may be used here. In someembodiments, the surfactant can be Tween 20™.

Mineral Coating

A scaffold, or portion or component thereof, described herein caninclude a surface modification or a coating. The mineral coating of ascaffold, as described herein, can be performed by any conventionalmanner. A mineral coating can be as described in U.S. application Ser.Nos. 13/407,441; 13/879,178; and 13/036,470 and are incorporated byreference.

The Examples describe exemplary methods for producing coated scaffoldsusing a mineral coating solution. For example, the mineral coatingsolution can be a modified simulated body fluid (mSBF). By adjusting themineral composition, and/or concentration of the mSBF, the compositionof the mineral precipitated on the scaffolds can be manipulated. Seealso U.S. Patent Application Publication US 2008/0095817 A1; U.S. Pat.No. 6,767,928 B1; U.S. Pat. No. 6,541,022 B1; PCT Publication WO2008/070355 A2; PCT Publication WO 2008/082766 A2; Murphy and Mooney,2001; Murphy and Messersmith, 2000.

As described herein, the mineral coating can be calcium-containing. Forexample the calcium-containing mineral coating can includehydroxyapatite (HAP), α-tricalcium phosphate (α-TCP), β-tricalciumphosphate (β-TCP), amorphous calcium phosphate, dicalcium phosphate,octacalcium phosphate, calcium phosphate (CaP), or calcium carbonate.The calcium-containing mineral coating can comprise a plurality oflayers, e.g., separate layers having distinct dissolution profiles.Under physiological conditions, solubility of calcium phosphate speciescan adhere to the following trend: amorphous calcium phosphate>dicalciumphosphate>octacalcium phosphate>β-TCP>HAP. A dicalcium phosphate mineralcan have a dissolution rate that is more than fifty times higher thanthat of HAP. Therefore, creation of a matrix with distinct calciumphosphate layers allows for a broad range of dissolution patterns.

For example, a mineral coating can be according to ISO 2337 ‘Implantsfor surgery—In vitro evaluation for apatite-forming ability of implantmaterials’. As another example, mineral coating can be an adaptedprotocol according to ISO 2337 ‘Implants for surgery—In vitro evaluationfor apatite-forming ability of implant materials’. As another example,the mineral coating can be performed by immersing a scaffold into amodified simulated body fluid at physiological conditions and continuousrotations. Continuous rotations can be replenishing the modifiedsimulated body fluid, replacing the modified simulated body fluid, orremoving and adding modified simulated body fluid.

As described herein, the scaffold can be incubated in modified simulatedbody fluid (mSBF) solutions to induce formation of a calciumphosphate-based mineral layer for mineral nucleation and growth. ThemSBF solution can contain ionic constituents of blood plasma, withdouble the concentrations of calcium and phosphate ions, held atphysiologic temperature and pH. The growth of calcium phosphate-basedminerals, specifically bone-like minerals, on bioresorbable polymermatrices using mSBF incubation has been demonstrated (Lin et al., 2004;Murphy et al., 2002, 2005).

As described herein, a mineral coating of a scaffold, as describedherein, can be performed by incubating a scaffold. For example, themineral coating, described herein, can be developed by incubating theconstituents in modified simulated body fluid (mSBF), for five days ormore at a pH of about 6.8 to about 7.4 and at a temperature of about 37°C. The SBF or mSBF can be refreshed daily. Using the chemicalcomposition described in the Examples, the procedure produces acalcium-deficient, carbonate-containing apatite material on alginate andon poly-(α-hydroxy esters). See U.S. Pat. No. 6,767,928, incorporatedherein by reference. mSBF can include elevated calcium and phosphate. Ingeneral, an increase in pH favors hydroxyapatite growth, while adecrease in pH favors octacalcium phosphate mineral growth.

As another example, conditions favorable for hydroxyapatite formationcan include a pH between about 5.0 and about 8.0 and a calciumconcentration multiplied by a phosphate concentration between about 10-5and about 10-8 M. Likewise, conditions favorable for octacalciumphosphate formation include a pH between about 6.0 and about 8.0 and acalcium concentration multiplied by a phosphate concentration betweenabout 10-5 and about 10-7.5 M. Furthermore, conditions favorable fordicalcium phosphate dehydrate formation can include a pH between about6.0 and about 8.0 and a calcium concentration multiplied by a phosphateconcentration between about 10-4 and about 10-6 M.

As another example, one could vary the pH of mSBF between about 5.0 andabout 6.0 to promote hydroxyapatite formation. Similarly, one could varythe pH of mSBF between about 6.0 and about 6.5 to promote octacalciumphosphate and hydroxyapatite formation. Likewise, one could vary the pHof mSBF between about 6.5 and about 8.0 to promote dicalcium phosphate,octacalcium phosphate, and hydroxyapatite formation.

As another example, the scaffold can be incubated for at least about 1day; at least about 2 days; at least about 3 days; at least about 4days; at least about 5 days; at least about 6 days; at least about 7days; at least about 8 days; at least about 9 days; at least about 10days; at least about 11 days; at least about 12 days; at least about 13days; at least about 14 days; at least about 15 days; at least about 16days; at least about 17 days; at least about 18 days; at least about 19days; at least about 20 days; at least about 21 days; at least about 22days; at least about 23 days; at least about 24 days; at least about 25days; at least about 26 days; at least about 27 days; at least about 28days; at least about 29 days; or at least about 30 days. It isunderstood that recitation of the above discrete values includes a rangebetween each recited value.

For example, the scaffold can be incubated for about 1 day; about 2days; about 3 days; about 4 days; about 5 days; about 6 days; about 7days; about 8 days; about 9 days; about 10 days; about 11 days; about 12days; about 13 days; about 14 days; about 15 days; about 16 days; about17 days; about 18 days; about 19 days; about 20 days; about 21 days;about 22 days; about 23 days; about 24 days; about 25 days; about 26days; about 27 days; about 28 days; about 29 days; or about 30 days. Itis understood that recitation of the above discrete values includes arange between each recited value.

A mineral coating of a scaffold, as described herein, can be performedby incubating a scaffold at a temperature. For example, the scaffold canbe incubated at a physiologically relevant temperature. As anotherexample, the scaffold can be incubated at a temperature of about 1° C.;about 2° C.; about 3° C.; about 4° C.; about 5° C.; about 6° C.; about7° C.; about 8° C.; about 9° C.; about 10° C.; about 11° C.; about 12°C.; about 13° C.; about 14° C.; about 15° C.; about 16° C.; about 17°C.; about 18° C.; about 19° C.; about 20° C.; about 21° C.; about 22°C.; about 23° C.; about 24° C.; about 25° C.; about 26° C.; about 27°C.; about 28° C.; about 29° C.; about 30° C.; about 31° C.; about 32°C.; about 33° C.; about 34° C.; about 35° C.; about 36° C.; about 37°C.; about 38° C.; about 39° C.; about 40° C.; about 41° C.; about 42°C.; about 43° C.; about 44° C.; about 45° C.; about 46° C.; about 47°C.; about 48° C.; about 49° C.; about 50° C.; about 51° C.; about 52°C.; about 53° C.; about 54° C.; about 55° C.; about 56° C.; about 57°C.; about 58° C.; about 59° C.; about 60° C.; about 61° C.; about 62°C.; about 63° C.; about 64° C.; about 65° C.; about 66° C.; about 67°C.; about 68° C.; about 69° C.; about 70° C.; about 71° C.; about 72°C.; about 73° C.; about 74° C.; about 75° C.; about 76° C.; about 77°C.; about 78° C.; about 79° C.; about 80° C.; about 81° C.; about 82°C.; about 83° C.; about 84° C.; about 85° C.; about 86° C.; about 87°C.; about 88° C.; about 89° C.; about 90° C.; about 91° C.; about 92°C.; about 93° C.; about 94° C.; about 95° C.; about 96° C.; about 97°C.; about 98° C.; about 99° C.; or about 100° C.

A scaffold, or portion or component thereof, can be coated individuallyor in groups using, for example, a CaP coating technology. A scaffold,or portion or component thereof, can be modified individually or ingroups using a technique such as aminolysis for RGD attachment, chemicalconjugation, layer by layer deposition, or chemical vapor deposition.

Prior to deposition of the first calcium-containing mineral, thescaffold may be surface-functionalized to allow increased mineraldeposition by utilizing chemical pre-treatment to achieve surfacehydrolysis (e.g., using an NaOH solution). Surface degradation by thistechnique can cause an increase in the amount of polar oxygen functionalgroups on the surface of the material.

The functionalized surface can then be incubated in a mineral-containingsolution (e.g., modified simulated body fluid). The mineral coatingprocess, as described herein, can mimic natural biomineralizationprocesses.

The mineral coating, as described herein, can be similar in structureand composition to human bone mineral. For example, the mineral coatingcan include spherical clusters with a plate-like structure or aplate-like structure and a carbonate-substituted, calcium deficienthydroxyapatite phase composition. As another example, the coating can bean osteoconductive mineral coating.

As another example, the mineral coating can include an apatite. Apatitecan include calcium phosphate, calcium carbonate, calcium fluoride,calcium hydroxide, or citrate.

As another example, a mineral coating can comprises a plurality ofdiscrete mineral islands on the scaffold, or the mineral coating can beformed on the entire surface of the scaffold. As another example, themineral coating can comprise a substantially homogeneous mineralcoating. In other embodiments, the mineral coatings can be any suitablecoating material containing calcium and phosphate, such ashydroxyapatite, calcium-deficient carbonate-containing hydroxyapatite,tricalcium phosphate, amorphous calcium phosphate, octacalciumphosphate, dicalcium phosphate, calcium phosphate, or a mixture thereof.For example, an osteoconductive mineral coating can be calcium-deficientcarbonate-containing hydroxyapatite.

As another example, the mineral coating can include hydroxyapatite.Calcium deficient hydroxyapatite can have a formula ofCa_(10-x)(PO₄)_(6-x)(HPO₄)_(x)(OH)_(2-x). Stoichiometric hydroxyapatitecan have a chemical formula of Ca₁₀(PO₄)₆(OH)₂ or can be also written asCa₅(PO₄)₃(OH). Hydroxyapatite can be predominantly crystalline, but maybe present in amorphous forms.

The mineral coating, as described herein, can include at least about 1hydroxyapatite. For example, the mineral coating can include at leastabout 1 hydroxyapatite; at least about 2% hydroxyapatite; at least about3% hydroxyapatite; at least about 4% hydroxyapatite; at least about 5%hydroxyapatite; at least about 6% hydroxyapatite; at least about 7%hydroxyapatite; at least about 8% hydroxyapatite; at least about 9%hydroxyapatite; at least about 10% hydroxyapatite; at least about 11hydroxyapatite; at least about 12% hydroxyapatite; at least about 13%hydroxyapatite; at least about 14% hydroxyapatite; at least about 15%hydroxyapatite; at least about 16% hydroxyapatite; at least about 17%hydroxyapatite; at least about 18% hydroxyapatite; at least about 19%hydroxyapatite; at least about 20% hydroxyapatite; at least about 21hydroxyapatite; at least about 22% hydroxyapatite; at least about 23%hydroxyapatite; at least about 24% hydroxyapatite; at least about 25%hydroxyapatite; at least about 26% hydroxyapatite; at least about 27%hydroxyapatite; at least about 28% hydroxyapatite; at least about 29%hydroxyapatite; at least about 30% hydroxyapatite; at least about 31%hydroxyapatite; at least about 32% hydroxyapatite; at least about 33%hydroxyapatite; at least about 34% hydroxyapatite; at least about 35%hydroxyapatite; at least about 36% hydroxyapatite; at least about 37%hydroxyapatite; at least about 38% hydroxyapatite; at least about 39%hydroxyapatite; at least about 40% hydroxyapatite; at least about 41hydroxyapatite; at least about 42% hydroxyapatite; at least about 43%hydroxyapatite; at least about 44% hydroxyapatite; at least about 45%hydroxyapatite; at least about 46% hydroxyapatite; at least about 47%hydroxyapatite; at least about 48% hydroxyapatite; at least about 49%hydroxyapatite; at least about 50% hydroxyapatite; at least about 51hydroxyapatite; at least about 52% hydroxyapatite; at least about 53%hydroxyapatite; at least about 54% hydroxyapatite; at least about 55%hydroxyapatite; at least about 56% hydroxyapatite; at least about 57%hydroxyapatite; at least about 58% hydroxyapatite; at least about 59%hydroxyapatite; at least about 60% hydroxyapatite; at least about 61hydroxyapatite; at least about 62% hydroxyapatite; at least about 63%hydroxyapatite; at least about 64% hydroxyapatite; at least about 65%hydroxyapatite; at least about 66% hydroxyapatite; at least about 67%hydroxyapatite; at least about 68% hydroxyapatite; at least about 69%hydroxyapatite; at least about 70% hydroxyapatite; at least about 71%hydroxyapatite; at least about 72% hydroxyapatite; at least about 73%hydroxyapatite; at least about 74% hydroxyapatite; at least about 75%hydroxyapatite; at least about 76% hydroxyapatite; at least about 77%hydroxyapatite; at least about 78% hydroxyapatite; at least about 79%hydroxyapatite; at least about 80% hydroxyapatite; at least about 81hydroxyapatite; at least about 82% hydroxyapatite; at least about 83%hydroxyapatite; at least about 84% hydroxyapatite; at least about 85%hydroxyapatite; at least about 86% hydroxyapatite; at least about 87%hydroxyapatite; at least about 88% hydroxyapatite; at least about 89%hydroxyapatite; at least about 90% hydroxyapatite; at least about 91hydroxyapatite; at least about 92% hydroxyapatite; at least about 93%hydroxyapatite; at least about 94% hydroxyapatite; at least about 95%hydroxyapatite; at least about 96% hydroxyapatite; at least about 97%hydroxyapatite; at least about 98% hydroxyapatite; at least about 99%hydroxyapatite; or at least about 100% hydroxyapatite. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

As another example, the mineral coating can include about 1%hydroxyapatite; about 2% hydroxyapatite; about 3% hydroxyapatite; about4% hydroxyapatite; about 5% hydroxyapatite; about 6% hydroxyapatite;about 7% hydroxyapatite; about 8% hydroxyapatite; about 9%hydroxyapatite; about 10% hydroxyapatite; about 11% hydroxyapatite;about 12% hydroxyapatite; about 13% hydroxyapatite; about 14%hydroxyapatite; about 15% hydroxyapatite; about 16% hydroxyapatite;about 17% hydroxyapatite; about 18% hydroxyapatite; about 19%hydroxyapatite; about 20% hydroxyapatite; about 21% hydroxyapatite;about 22% hydroxyapatite; about 23% hydroxyapatite; about 24%hydroxyapatite; about 25% hydroxyapatite; about 26% hydroxyapatite;about 27% hydroxyapatite; about 28% hydroxyapatite; about 29%hydroxyapatite; about 30% hydroxyapatite; about 31% hydroxyapatite;about 32% hydroxyapatite; about 33% hydroxyapatite; about 34%hydroxyapatite; about 35% hydroxyapatite; about 36% hydroxyapatite;about 37% hydroxyapatite; about 38% hydroxyapatite; about 39%hydroxyapatite; about 40% hydroxyapatite; about 41% hydroxyapatite;about 42% hydroxyapatite; about 43% hydroxyapatite; about 44%hydroxyapatite; about 45% hydroxyapatite; about 46% hydroxyapatite;about 47% hydroxyapatite; about 48% hydroxyapatite; about 49%hydroxyapatite; about 50% hydroxyapatite; about 51% hydroxyapatite;about 52% hydroxyapatite; about 53% hydroxyapatite; about 54%hydroxyapatite; about 55% hydroxyapatite; about 56% hydroxyapatite;about 57% hydroxyapatite; about 58% hydroxyapatite; about 59%hydroxyapatite; about 60% hydroxyapatite; about 61% hydroxyapatite;about 62% hydroxyapatite; about 63% hydroxyapatite; about 64%hydroxyapatite; about 65% hydroxyapatite; about 66% hydroxyapatite;about 67% hydroxyapatite; about 68% hydroxyapatite; about 69%hydroxyapatite; about 70% hydroxyapatite; about 71% hydroxyapatite;about 72% hydroxyapatite; about 73% hydroxyapatite; about 74%hydroxyapatite; about 75% hydroxyapatite; about 76% hydroxyapatite;about 77% hydroxyapatite; about 78% hydroxyapatite; about 79%hydroxyapatite; about 80% hydroxyapatite; about 81% hydroxyapatite;about 82% hydroxyapatite; about 83% hydroxyapatite; about 84%hydroxyapatite; about 85% hydroxyapatite; about 86% hydroxyapatite;about 87% hydroxyapatite; about 88% hydroxyapatite; about 89%hydroxyapatite; about 90% hydroxyapatite; about 91% hydroxyapatite;about 92% hydroxyapatite; about 93% hydroxyapatite; about 94%hydroxyapatite; about 95% hydroxyapatite; about 96% hydroxyapatite;about 97% hydroxyapatite; about 98% hydroxyapatite; about 99%hydroxyapatite; or about 100% hydroxyapatite. It is understood thatrecitation of the above discrete values includes a range between eachrecited value.

The mineral coating, as described herein, can include octacalciumphosphate. Octacalcium phosphate has a chemical formula ofCa₈H₂(PO₄)₆.5H₂O or can also be written as Ca₄HO₁₂P₃. Octacalciumphosphate has been shown to be a precursor of hydroxyapatite. Hydrolysisof Octacalcium phosphate can create hydroxyapatite. Octacalciumphosphate can be predominantly crystalline, but may be present inamorphous forms.

The mineral coating, as described herein, can include at least about 1octacalcium phosphate. For example, the mineral coating can include atleast about 1% octacalcium phosphate; at least about 2% octacalciumphosphate; at least about 3% octacalcium phosphate; at least about 4%octacalcium phosphate; at least about 5% octacalcium phosphate; at leastabout 6% octacalcium phosphate; at least about 7% octacalcium phosphate;at least about 8% octacalcium phosphate; at least about 9% octacalciumphosphate; at least about 10% octacalcium phosphate; at least about 11%octacalcium phosphate; at least about 12% octacalcium phosphate; atleast about 13% octacalcium phosphate; at least about 14% octacalciumphosphate; at least about 15% octacalcium phosphate; at least about 16%octacalcium phosphate; at least about 17% octacalcium phosphate; atleast about 18% octacalcium phosphate; at least about 19% octacalciumphosphate; at least about 20% octacalcium phosphate; at least about 21%octacalcium phosphate; at least about 22% octacalcium phosphate; atleast about 23% octacalcium phosphate; at least about 24% octacalciumphosphate; at least about 25% octacalcium phosphate; at least about 26%octacalcium phosphate; at least about 27% octacalcium phosphate; atleast about 28% octacalcium phosphate; at least about 29% octacalciumphosphate; at least about 30% octacalcium phosphate; at least about 31%octacalcium phosphate; at least about 32% octacalcium phosphate; atleast about 33% octacalcium phosphate; at least about 34% octacalciumphosphate; at least about 35% octacalcium phosphate; at least about 36%octacalcium phosphate; at least about 37% octacalcium phosphate; atleast about 38% octacalcium phosphate; at least about 39% octacalciumphosphate; at least about 40% octacalcium phosphate; at least about 41%octacalcium phosphate; at least about 42% octacalcium phosphate; atleast about 43% octacalcium phosphate; at least about 44% octacalciumphosphate; at least about 45% octacalcium phosphate; at least about 46%octacalcium phosphate; at least about 47% octacalcium phosphate; atleast about 48% octacalcium phosphate; at least about 49% octacalciumphosphate; at least about 50% octacalcium phosphate; at least about 51%octacalcium phosphate; at least about 52% octacalcium phosphate; atleast about 53% octacalcium phosphate; at least about 54% octacalciumphosphate; at least about 55% octacalcium phosphate; at least about 56%octacalcium phosphate; at least about 57% octacalcium phosphate; atleast about 58% octacalcium phosphate; at least about 59% octacalciumphosphate; at least about 60% octacalcium phosphate; at least about 61%octacalcium phosphate; at least about 62% octacalcium phosphate; atleast about 63% octacalcium phosphate; at least about 64% octacalciumphosphate; at least about 65% octacalcium phosphate; at least about 66%octacalcium phosphate; at least about 67% octacalcium phosphate; atleast about 68% octacalcium phosphate; at least about 69% octacalciumphosphate; at least about 70% octacalcium phosphate; at least about 71octacalcium phosphate; at least about 72% octacalcium phosphate; atleast about 73% octacalcium phosphate; at least about 74% octacalciumphosphate; at least about 75% octacalcium phosphate; at least about 76%octacalcium phosphate; at least about 77% octacalcium phosphate; atleast about 78% octacalcium phosphate; at least about 79% octacalciumphosphate; at least about 80% octacalcium phosphate; at least about 81%octacalcium phosphate; at least about 82% octacalcium phosphate; atleast about 83% octacalcium phosphate; at least about 84% octacalciumphosphate; at least about 85% octacalcium phosphate; at least about 86%octacalcium phosphate; at least about 87% octacalcium phosphate; atleast about 88% octacalcium phosphate; at least about 89% octacalciumphosphate; at least about 90% octacalcium phosphate; at least about 91%octacalcium phosphate; at least about 92% octacalcium phosphate; atleast about 93% octacalcium phosphate; at least about 94% octacalciumphosphate; at least about 95% octacalcium phosphate; at least about 96%octacalcium phosphate; at least about 97% octacalcium phosphate; atleast about 98% octacalcium phosphate; at least about 99% octacalciumphosphate; or at least about 100% octacalcium phosphate. It isunderstood that recitation of the above discrete values includes a rangebetween each recited value.

As another example, the mineral coating can include about 1% octacalciumphosphate; about 2% octacalcium phosphate; about 3% octacalciumphosphate; about 4% octacalcium phosphate; about 5% octacalciumphosphate; about 6% octacalcium phosphate; about 7% octacalciumphosphate; about 8% octacalcium phosphate; about 9% octacalciumphosphate; about 10% octacalcium phosphate; about 11% octacalciumphosphate; about 12% octacalcium phosphate; about 13% octacalciumphosphate; about 14% octacalcium phosphate; about 15% octacalciumphosphate; about 16% octacalcium phosphate; about 17% octacalciumphosphate; about 18% octacalcium phosphate; about 19% octacalciumphosphate; about 20% octacalcium phosphate; about 21% octacalciumphosphate; about 22% octacalcium phosphate; about 23% octacalciumphosphate; about 24% octacalcium phosphate; about 25% octacalciumphosphate; about 26% octacalcium phosphate; about 27% octacalciumphosphate; about 28% octacalcium phosphate; about 29% octacalciumphosphate; about 30% octacalcium phosphate; about 31% octacalciumphosphate; about 32% octacalcium phosphate; about 33% octacalciumphosphate; about 34% octacalcium phosphate; about 35% octacalciumphosphate; about 36% octacalcium phosphate; about 37% octacalciumphosphate; about 38% octacalcium phosphate; about 39% octacalciumphosphate; about 40% octacalcium phosphate; about 41% octacalciumphosphate; about 42% octacalcium phosphate; about 43% octacalciumphosphate; about 44% octacalcium phosphate; about 45% octacalciumphosphate; about 46% octacalcium phosphate; about 47% octacalciumphosphate; about 48% octacalcium phosphate; about 49% octacalciumphosphate; about 50% octacalcium phosphate; about 51% octacalciumphosphate; about 52% octacalcium phosphate; about 53% octacalciumphosphate; about 54% octacalcium phosphate; about 55% octacalciumphosphate; about 56% octacalcium phosphate; about 57% octacalciumphosphate; about 58% octacalcium phosphate; about 59% octacalciumphosphate; about 60% octacalcium phosphate; about 61% octacalciumphosphate; about 62% octacalcium phosphate; about 63% octacalciumphosphate; about 64% octacalcium phosphate; about 65% octacalciumphosphate; about 66% octacalcium phosphate; about 67% octacalciumphosphate; about 68% octacalcium phosphate; about 69% octacalciumphosphate; about 70% octacalcium phosphate; about 71% octacalciumphosphate; about 72% octacalcium phosphate; about 73% octacalciumphosphate; about 74% octacalcium phosphate; about 75% octacalciumphosphate; about 76% octacalcium phosphate; about 77% octacalciumphosphate; about 78% octacalcium phosphate; about 79% octacalciumphosphate; about 80% octacalcium phosphate; about 81% octacalciumphosphate; about 82% octacalcium phosphate; about 83% octacalciumphosphate; about 84% octacalcium phosphate; about 85% octacalciumphosphate; about 86% octacalcium phosphate; about 87% octacalciumphosphate; about 88% octacalcium phosphate; about 89% octacalciumphosphate; about 90% octacalcium phosphate; about 91 octacalciumphosphate; about 92% octacalcium phosphate; about 93% octacalciumphosphate; about 94% octacalcium phosphate; about 95% octacalciumphosphate; about 96% octacalcium phosphate; about 97% octacalciumphosphate; about 98% octacalcium phosphate; about 99% octacalciumphosphate; or about 100% octacalcium phosphate. It is understood thatrecitation of the above discrete values includes a range between eachrecited value.

The mineral coating, as described herein, can include at least about 1%porosity. For example, the mineral coating, as described herein, caninclude a porosity of at least about 1% porosity; at least about 2%porosity; at least about 3% porosity; at least about 4% porosity; atleast about 5% porosity; at least about 6% porosity; at least about 7%porosity; at least about 8% porosity; at least about 9% porosity; atleast about 10% porosity; at least about 11% porosity; at least about12% porosity; at least about 13% porosity; at least about 14% porosity;at least about 15% porosity; at least about 16% porosity; at least about17% porosity; at least about 18% porosity; at least about 19% porosity;at least about 20% porosity; at least about 21% porosity; at least about22% porosity; at least about 23% porosity; at least about 24% porosity;at least about 25% porosity; at least about 26% porosity; at least about27% porosity; at least about 28% porosity; at least about 29% porosity;at least about 30% porosity; at least about 31% porosity; at least about32% porosity; at least about 33% porosity; at least about 34% porosity;at least about 35% porosity; at least about 36% porosity; at least about37% porosity; at least about 38% porosity; at least about 39% porosity;at least about 40% porosity; at least about 41% porosity; at least about42% porosity; at least about 43% porosity; at least about 44% porosity;at least about 45% porosity; at least about 46% porosity; at least about47% porosity; at least about 48% porosity; at least about 49% porosity;at least about 50% porosity; at least about 51% porosity; at least about52% porosity; at least about 53% porosity; at least about 54% porosity;at least about 55% porosity; at least about 56% porosity; at least about57% porosity; at least about 58% porosity; at least about 59% porosity;at least about 60% porosity; at least about 61% porosity; at least about62% porosity; at least about 63% porosity; at least about 64% porosity;at least about 65% porosity; at least about 66% porosity; at least about67% porosity; at least about 68% porosity; at least about 69% porosity;at least about 70% porosity; at least about 71% porosity; at least about72% porosity; at least about 73% porosity; at least about 74% porosity;at least about 75% porosity; at least about 76% porosity; at least about77% porosity; at least about 78% porosity; at least about 79% porosity;at least about 80% porosity; at least about 81% porosity; at least about82% porosity; at least about 83% porosity; at least about 84% porosity;at least about 85% porosity; at least about 86% porosity; at least about87% porosity; at least about 88% porosity; at least about 89% porosity;at least about 90% porosity; at least about 91% porosity; at least about92% porosity; at least about 93% porosity; at least about 94% porosity;at least about 95% porosity; at least about 96% porosity; at least about97% porosity; at least about 98% porosity; at least about 99% porosity;or at least about 100% porosity. It is understood that recitation of theabove discrete values includes a range between each recited value.

As another example, the mineral coating can include about 1% porosity;about 2% porosity; about 3% porosity; about 4% porosity; about 5%porosity; about 6% porosity; about 7% porosity; about 8% porosity; about9% porosity; about 10% porosity; about 11% porosity; about 12% porosity;about 13% porosity; about 14% porosity; about 15% porosity; about 16%porosity; about 17% porosity; about 18% porosity; about 19% porosity;about 20% porosity; about 21% porosity; about 22% porosity; about 23%porosity; about 24% porosity; about 25% porosity; about 26% porosity;about 27% porosity; about 28% porosity; about 29% porosity; about 30%porosity; about 31% porosity; about 32% porosity; about 33% porosity;about 34% porosity; about 35% porosity; about 36% porosity; about 37%porosity; about 38% porosity; about 39% porosity; about 40% porosity;about 41% porosity; about 42% porosity; about 43% porosity; about 44%porosity; about 45% porosity; about 46% porosity; about 47% porosity;about 48% porosity; about 49% porosity; about 50% porosity; about 51%porosity; about 52% porosity; about 53% porosity; about 54% porosity;about 55% porosity; about 56% porosity; about 57% porosity; about 58%porosity; about 59% porosity; about 60% porosity; about 61% porosity;about 62% porosity; about 63% porosity; about 64% porosity; about 65%porosity; about 66% porosity; about 67% porosity; about 68% porosity;about 69% porosity; about 70% porosity; about 71% porosity; about 72%porosity; about 73% porosity; about 74% porosity; about 75% porosity;about 76% porosity; about 77% porosity; about 78% porosity; about 79%porosity; about 80% porosity; about 81% porosity; about 82% porosity;about 83% porosity; about 84% porosity; about 85% porosity; about 86%porosity; about 87% porosity; about 88% porosity; about 89% porosity;about 90% porosity; about 91% A porosity; about 92% porosity; about 93%porosity; about 94% porosity; about 95% porosity; about 96% porosity;about 97% porosity; about 98% porosity; about 99% porosity; or about100% porosity. It is understood that recitation of the above discretevalues includes a range between each recited value.

The mineral coating, as described herein, can include a pore diameterbetween about 1 nm and about 3500 nm. As another example, the mineralcoating, as described herein, can include a pore diameter between about100 and about 350 nm. As another example, the mineral coating, asdescribed herein, can include at least about 1 nm pore diameter; atleast about 10 nm pore diameter; at least about 20 nm pore diameter; atleast about 30 nm pore diameter; at least about 40 nm pore diameter; atleast about 50 nm pore diameter; at least about 55 nm pore diameter; atleast about 60 nm pore diameter; at least about 65 nm pore diameter; atleast about 70 nm pore diameter; at least about 75 nm pore diameter; atleast about 80 nm pore diameter; at least about 85 nm pore diameter; atleast about 90 nm pore diameter; at least about 95 nm pore diameter; atleast about 100 nm pore diameter; at least about 105 nm pore diameter;at least about 110 nm pore diameter; at least about 115 nm porediameter; at least about 120 nm pore diameter; at least about 125 nmpore diameter; at least about 130 nm pore diameter; at least about 140nm pore diameter; at least about 145 nm pore diameter; at least about150 nm pore diameter; at least about 155 nm pore diameter; at leastabout 160 nm pore diameter; at least about 165 nm pore diameter; atleast about 170 nm pore diameter; at least about 175 nm pore diameter;at least about 180 nm pore diameter; at least about 185 nm porediameter; at least about 190 nm pore diameter; at least about 200 nmpore diameter; at least about 205 nm pore diameter; at least about 210nm pore diameter; at least about 215 nm pore diameter; at least about220 nm pore diameter; at least about 225 nm pore diameter; at leastabout 230 nm pore diameter; at least about 235 nm pore diameter; atleast about 240 nm pore diameter; at least about 245 nm pore diameter;at least about 250 nm pore diameter; at least about 255 nm porediameter; at least about 260 nm pore diameter; at least about 265 nmpore diameter; at least about 270 nm pore diameter; at least about 275nm pore diameter; at least about 280 nm pore diameter; at least about285 nm pore diameter; at least about 290 nm pore diameter; at leastabout 295 nm pore diameter; at least about 300 nm pore diameter; atleast about 305 nm pore diameter; at least about 310 nm pore diameter;at least about 315 nm pore diameter; at least about 330 nm porediameter; at least about 335 nm pore diameter; at least about 340 nmpore diameter; at least about 345 nm pore diameter; at least about 350nm pore diameter; at least about 355 nm pore diameter; at least about360 nm pore diameter; at least about 365 nm pore diameter; at leastabout 370 nm pore diameter; at least about 375 nm pore diameter; atleast about 400 nm pore diameter; at least about 410 nm pore diameter;at least about 420 nm pore diameter; at least about 430 nm porediameter; at least about 440 nm pore diameter; at least about 450 nmpore diameter; at least about 460 nm pore diameter; at least about 470nm pore diameter; at least about 480 nm pore diameter; at least about490 nm pore diameter; at least about 500 nm pore diameter; at leastabout 600 nm pore diameter; at least about 700 nm pore diameter; atleast about 800 nm pore diameter; at least about 900 nm pore diameter;at least about 1000 nm pore diameter; at least about 1100 nm porediameter; at least about 1200 nm pore diameter; at least about 1300 nmpore diameter; at least about 1400 nm pore diameter; at least about 1500nm pore diameter; at least about 1600 nm pore diameter; at least about1700 nm pore diameter; at least about 1800 nm pore diameter; at leastabout 1900 nm pore diameter; at least about 2000 nm pore diameter; atleast about 2100 nm pore diameter; at least about 2200 nm pore diameter;at least about 2300 nm pore diameter; at least about 2400 nm porediameter; at least about 2500 nm pore diameter; at least about 2600 nmpore diameter; at least about 2700 nm pore diameter; at least about 2800nm pore diameter; at least about 2900 nm pore diameter; at least about3000 nm pore diameter; at least about 3100 nm pore diameter; at leastabout 3200 nm pore diameter; at least about 3300 nm pore diameter; atleast about 3400 nm pore diameter; or at least about 3500 nm porediameter. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

As another example, the mineral coating, as described herein, caninclude about 1 nm pore diameter; about 10 nm pore diameter; about 20 nmpore diameter; about 30 nm pore diameter; about 40 nm pore diameter;about 50 nm pore diameter; about 55 nm pore diameter; about 60 nm porediameter; about 65 nm pore diameter; about 70 nm pore diameter; about 75nm pore diameter; about 80 nm pore diameter; about 85 nm pore diameter;about 90 nm pore diameter; about 95 nm pore diameter; about 100 nm porediameter; about 105 nm pore diameter; about 110 nm pore diameter; about115 nm pore diameter; about 120 nm pore diameter; about 125 nm porediameter; about 130 nm pore diameter; about 140 nm pore diameter; about145 nm pore diameter; about 150 nm pore diameter; about 155 nm porediameter; about 160 nm pore diameter; about 165 nm pore diameter; about170 nm pore diameter; about 175 nm pore diameter; about 180 nm porediameter; about 185 nm pore diameter; about 190 nm pore diameter; about200 nm pore diameter; about 205 nm pore diameter; about 210 nm porediameter; about 215 nm pore diameter; about 220 nm pore diameter; about225 nm pore diameter; about 230 nm pore diameter; about 235 nm porediameter; about 240 nm pore diameter; about 245 nm pore diameter; about250 nm pore diameter; about 255 nm pore diameter; about 260 nm porediameter; about 265 nm pore diameter; about 270 nm pore diameter; about275 nm pore diameter; about 280 nm pore diameter; about 285 nm porediameter; about 290 nm pore diameter; about 295 nm pore diameter; about300 nm pore diameter; about 305 nm pore diameter; about 310 nm porediameter; about 315 nm pore diameter; about 330 nm pore diameter; about335 nm pore diameter; about 340 nm pore diameter; about 345 nm porediameter; about 350 nm pore diameter; about 355 nm pore diameter; about360 nm pore diameter; about 365 nm pore diameter; about 370 nm porediameter; about 375 nm pore diameter; about 400 nm pore diameter; about410 nm pore diameter; about 420 nm pore diameter; about 430 nm porediameter; about 440 nm pore diameter; about 450 nm pore diameter; about460 nm pore diameter; about 470 nm pore diameter; about 480 nm porediameter; about 490 nm pore diameter; about 500 nm pore diameter; about600 nm pore diameter; about 700 nm pore diameter; about 800 nm porediameter; about 900 nm pore diameter; about 1000 nm pore diameter; about1100 nm pore diameter; about 1200 nm pore diameter; about 1300 nm porediameter; about 1400 nm pore diameter; about 1500 nm pore diameter;about 1600 nm pore diameter; about 1700 nm pore diameter; about 1800 nmpore diameter; about 1900 nm pore diameter; about 2000 nm pore diameter;about 2100 nm pore diameter; about 2200 nm pore diameter; about 2300 nmpore diameter; about 2400 nm pore diameter; about 2500 nm pore diameter;about 2600 nm pore diameter; about 2700 nm pore diameter; about 2800 nmpore diameter; about 2900 nm pore diameter; about 3000 nm pore diameter;about 3100 nm pore diameter; about 3200 nm pore diameter; about 3300 nmpore diameter; about 3400 nm pore diameter; or about 3500 nm porediameter. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

The mineral coating, as described herein, can include a ratio of atleast about 0.1 Ca/P. For example, the mineral coating can include aratio of at least about 0.1 Ca/P; at least about 0.2 Ca/P; at leastabout 0.3 Ca/P; at least about 0.4 Ca/P; at least about 0.5 Ca/P; atleast about 0.6 Ca/P; at least about 0.7 Ca/P; at least about 0.8 Ca/P;at least about 0.9 Ca/P; at least about 1.0 Ca/P; at least about 1.1Ca/P; at least about 1.2 Ca/P; at least about 1.3 Ca/P; at least about1.4 Ca/P; at least about 1.5 Ca/P; at least about 1.6 Ca/P; at leastabout 1.7 Ca/P; at least about 1.8 Ca/P; at least about 1.9 Ca/P; atleast about 2.0 Ca/P; at least about 2.1 Ca/P; at least about 2.2 Ca/P;at least about 2.3 Ca/P; at least about 2.4 Ca/P; at least about 2.5Ca/P; at least about 2.6 Ca/P; at least about 2.7 Ca/P; at least about2.8 Ca/P; at least about 2.9 Ca/P; at least about 3.0 Ca/P; at leastabout 3.1 Ca/P; at least about 3.2 Ca/P; at least about 3.3 Ca/P; atleast about 3.4 Ca/P; at least about 3.5 Ca/P; at least about 3.6 Ca/P;at least about 3.7 Ca/P; at least about 3.8 Ca/P; at least about 3.9Ca/P; at least about 4 Ca/P; at least about 5 Ca/P; at least about 6Ca/P; at least about 7 Ca/P; at least about 8 Ca/P; at least about 9Ca/P; at least about 10 Ca/P; at least about 11 Ca/P; at least about 12Ca/P; at least about 13 Ca/P; at least about 14 Ca/P; at least about 15Ca/P; at least about 16 Ca/P; at least about 17 Ca/P; at least about 18Ca/P; at least about 19 Ca/P; or at least about 20 Ca/P. It isunderstood that recitation of the above discrete values includes a rangebetween each recited value.

As another example, the mineral coating can include a ratio of about 0.1Ca/P; about 0.2 Ca/P; about 0.3 Ca/P; about 0.4 Ca/P; about 0.5 Ca/P;about 0.6 Ca/P; about 0.7 Ca/P; about 0.8 Ca/P; about 0.9 Ca/P; about1.0 Ca/P; about 1.1 Ca/P; about 1.2 Ca/P; about 1.3 Ca/P; about 1.4Ca/P; about 1.5 Ca/P; about 1.6 Ca/P; about 1.7 Ca/P; about 1.8 Ca/P;about 1.9 Ca/P; about 2.0 Ca/P; about 2.1 Ca/P; about 2.2 Ca/P; about2.3 Ca/P; about 2.4 Ca/P; about 2.5 Ca/P; about 2.6 Ca/P; about 2.7Ca/P; about 2.8 Ca/P; about 2.9 Ca/P; about 3.0 Ca/P; about 3.1 Ca/P;about 3.2 Ca/P; about 3.3 Ca/P; about 3.4 Ca/P; about 3.5 Ca/P; about3.6 Ca/P; about 3.7 Ca/P; about 3.8 Ca/P; about 3.9 Ca/P; about 4 Ca/P;about 5 Ca/P; about 6 Ca/P; about 7 Ca/P; about 8 Ca/P; about 9 Ca/P;about 10 Ca/P; about 11 Ca/P; about 12 Ca/P; about 13 Ca/P; about 14Ca/P; about 15 Ca/P; about 16 Ca/P; about 17 Ca/P; about 18 Ca/P; about19 Ca/P; or about 20 Ca/P. It is understood that recitation of the abovediscrete values includes a range between each recited value.

A mineral coating, as described herein, can be characterized byconventional methods. For example, mineral formation in mSBF can betracked by analyzing changes in solution calcium concentration using acalcium sensitive electrode (Denver Instrument, Denver, Colo.). Aftertheir growth, the mineral matrices can be dissolved and analyzed forcalcium and phosphate ion content to quantify mineral formation, and themineral crystals can be analyzed morphologically and compositionallyusing a scanning electron microscope (SEM), e.g., with a Noran SiLidetector for elemental analysis.

For example, the crystalline phase can be characterized by X-raydiffraction, where 28 is in the range of 15-35° or 25.8°, 28.1°, 28.9°,31.8°, and 32.1°.

As another example, as described herein, the chemical composition orcrystalline phase can be characterized by Fourier transform infraredspectroscopy (FTIR), where carbonate peaks can be in the 1400-1500 cm⁻¹region and phosphate peaks can be in the 900-1100 cm⁻¹ region or about570 cm⁻¹, 962 cm⁻¹, and 1050 cm⁻¹.

As another example, as described herein, dissolution of mineral layerscan also be characterized by measuring release of calcium and phosphateions during incubation in tris-buffered saline at physiologicallyrelevant conditions (e.g., 37° C., pH 7.4).

As another example, as described herein, calcium and phosphateconcentrations can be measured using previously described colorimetricassays (see Murphy et al., “Bioinspired growth of crystalline carbonateapatite on biodegradable polymer substrata”, J Am Chem Soc 124:1910-7,2002). Each of the characterization methods described herein are routinein analysis of inorganic materials, and is consistent with FDA's goodguidance practices for design and testing of calcium phosphate coatings(see Devices FDoGaR. Calcium phosphate coating draft guidance forpreparation of FDA submissions for orthopedic and dental endosseousimplants. 1997).

As another example, as described herein, the mineral coating, can bepredominantly crystalline, but can be present in amorphous forms. Forexample, the mineral coating can have at least about 5% crystallinity.For example, a mineral coating can include at least about 5%crystallinity; at least about 10% crystallinity; at least about 15%crystallinity; at least about 20% crystallinity; at least about 25%crystallinity; at least about 30% crystallinity; at least about 40%crystallinity; at least about 45% crystallinity; at least about 46%crystallinity; at least about 47% crystallinity; at least about 48%crystallinity; at least about 49% crystallinity; at least about 50%crystallinity; at least about 51% crystallinity; at least about 52%crystallinity; at least about 53% crystallinity; at least about 54%crystallinity; at least about 55% crystallinity; at least about 56%crystallinity; at least about 57% crystallinity; at least about 58%crystallinity; at least about 59% crystallinity; at least about 60%crystallinity; at least about 61% crystallinity; at least about 62%crystallinity; at least about 63% crystallinity; at least about 64%crystallinity; at least about 65% crystallinity; at least about 66%crystallinity; at least about 67% crystallinity; at least about 68%crystallinity; at least about 69% crystallinity; at least about 70%crystallinity; at least about 71% crystallinity; at least about 72%crystallinity; at least about 73% crystallinity; at least about 74%crystallinity; at least about 75% crystallinity; at least about 76%crystallinity; at least about 77% crystallinity; at least about 78%crystallinity; at least about 79% crystallinity; at least about 80%crystallinity; at least about 81% crystallinity; at least about 82%crystallinity; at least about 83% crystallinity; at least about 84%crystallinity; at least about 85% crystallinity; at least about 86%crystallinity; at least about 87% crystallinity; at least about 88%crystallinity; at least about 89% crystallinity; at least about 90%crystallinity; at least about 91% crystallinity; at least about 92%crystallinity; at least about 93% crystallinity; at least about 94%crystallinity; at least about 95% crystallinity; at least about 96%crystallinity; at least about 97% crystallinity; at least about 98%crystallinity; at least about 99% crystallinity; or at least about 100%crystallinity. It is understood that recitation of the above discretevalues includes a range between each recited value.

As another example, a mineral coating can include about 5%crystallinity; 10% crystallinity; about 15% crystallinity; about 20%crystallinity; about 25% crystallinity; about 30% crystallinity; about40% crystallinity; about 45% crystallinity; about 46% crystallinity;about 47% crystallinity; about 48% crystallinity; about 49%crystallinity; about 50% crystallinity; about 51% crystallinity; about52% crystallinity; about 53% crystallinity; about 54% crystallinity;about 55% crystallinity; about 56% crystallinity; about 57%crystallinity; about 58% crystallinity; about 59% crystallinity; about60% crystallinity; about 61% crystallinity; about 62% crystallinity;about 63% crystallinity; about 64% crystallinity; about 65%crystallinity; about 66% crystallinity; about 67% crystallinity; about68% crystallinity; about 69% crystallinity; about 70% crystallinity;about 71% crystallinity; about 72% crystallinity; about 73%crystallinity; about 74% crystallinity; about 75% crystallinity; about76% crystallinity; about 77% crystallinity; about 78% crystallinity;about 79% crystallinity; about 80% crystallinity; about 81%crystallinity; about 82% crystallinity; about 83% crystallinity; about84% crystallinity; about 85% crystallinity; about 86% crystallinity;about 87% crystallinity; about 88% crystallinity; about 89%crystallinity; about 90% crystallinity; about 91% crystallinity; about92% crystallinity; about 93% crystallinity; about 94% crystallinity;about 95% crystallinity; about 96% crystallinity; about 97%crystallinity; about 98% crystallinity; about 99% crystallinity; orabout 100% crystallinity. It is understood that recitation of the abovediscrete values includes a range between each recited value.

As described herein, osteoconductivity and osteoinductivity can beconferred to scaffolds (e.g., orthopedic implant materials) usingcalcium phosphate coatings. Based on the well-defined osteoconductivityand potential osteoinductivity of calcium-phosphate-based mineralcoatings, calcium phosphate mineral growth can be advantageouslyutilized to coat scaffolds.

Auxiliary Components

The mineral coating, as described herein, can include auxiliarycomponents. For example, the auxiliary components can be incorporatedonto the scaffold, the surface of the coating, or within the coating ofthe scaffold.

For example, an auxiliary component can provide desirablecharacteristics to the mineral coating, such as improvedosteoconductivity, osteoinductivity, osteopromotion, osteogenesis,strength, antibacterial, antimicrobial properties, biostatic, oranti-infection properties.

As described herein, an auxiliary component can be an organic orcollagen matrix, such as demineralized bone matrix (DBM). As describedherein, DBM can be of any form known in the art. For example DBM can bea demineralized bone powder, demineralized bone extract, demineralizedbone gelatin, granules, fragments, pellets, slices, shavings, putty,paste, mix, or strips.

As described herein, DBM can be incorporated into or onto the coating orscaffold by any method known in the art. As another example, DBM can beloaded or integrated into a porous material, such as a porous scaffold.As another example, DBM can be loaded into a medical device, such as aporous, coated, bioresorbable implant formed via 3-D printing. Asanother example, DBM can be combined with a mineral-coated device. Asanother example, DBM can be incorporated with the mineral coating,scaffold, or matrix material by mixing, coating, or loading.

As described herein DBM can be integrated with the scaffold or mineralcoated scaffold by any method known in the art. The incorporation of DBMinto a mineral coating can be as described in Ozturk et al. 2006 IntOrth 30, 147-152 or US Pat Pub No. 2008/0233203.

As described herein, preparing DBM to be integrated with a scaffold cancomprise mixing DBM with an aqueous solution. For example, the aqueoussolution can be the mineral coating solution as described herein. Asanother example, the aqueous solution can be a weak acid or guanidinehydrochloride. The mixture can be constantly agitated for a set amountof time to produce an aqueous demineralized bone extract. The extractcan then be filtered to remove any remaining solids, the acidneutralized or removed, and the extract used to coat the porousscaffold.

As described herein, the amount of DBM in a solution can be from about 1g to about 99 g DBM or from about 2 g to about 10 g DBM per 100 g ofaqueous solution. The amount of DBM can comprise at least about 1 g DBMper 100 g aqueous solution. For example, the amount of DBM in a solutioncan comprise at least about 1 g DBM per 100 g aqueous solution; at leastabout 2 g DBM per 100 g aqueous solution; at least about 3 g DBM per 100g aqueous solution; at least about 4 g DBM per 100 g aqueous solution;at least about 5 g DBM per 100 g aqueous solution; at least about 6 gDBM per 100 g aqueous solution; at least about 7 g DBM per 100 g aqueoussolution; at least about 8 g DBM per 100 g aqueous solution; at leastabout 9 g DBM per 100 g aqueous solution; at least about 10 g DBM per100 g aqueous solution; at least about 11 g DBM per 100 g aqueoussolution; at least about 12 g DBM per 100 g aqueous solution; at leastabout 13 g DBM per 100 g aqueous solution; at least about 14 g DBM per100 g aqueous solution; at least about 15 g DBM per 100 g aqueoussolution; at least about 16 g DBM per 100 g aqueous solution; at leastabout 17 g DBM per 100 g aqueous solution; at least about 18 g DBM per100 g aqueous solution; at least about 19 g DBM per 100 g aqueoussolution; at least about 20 g DBM per 100 g aqueous solution; at leastabout 21 g DBM per 100 g aqueous solution; at least about 22 g DBM per100 g aqueous solution; at least about 23 g DBM per 100 g aqueoussolution; at least about 24 g DBM per 100 g aqueous solution; at leastabout 25 g DBM per 100 g aqueous solution; at least about 26 g DBM per100 g aqueous solution; at least about 27 g DBM per 100 g aqueoussolution; at least about 28 g DBM per 100 g aqueous solution; at leastabout 29 g DBM per 100 g aqueous solution; at least about 30 g DBM per100 g aqueous solution; at least about 31 g DBM per 100 g aqueoussolution; at least about 32 g DBM per 100 g aqueous solution; at leastabout 33 g DBM per 100 g aqueous solution; at least about 34 g DBM per100 g aqueous solution; at least about 35 g DBM per 100 g aqueoussolution; at least about 36 g DBM per 100 g aqueous solution; at leastabout 37 g DBM per 100 g aqueous solution; at least about 38 g DBM per100 g aqueous solution; at least about 39 g DBM per 100 g aqueoussolution; at least about 40 g DBM per 100 g aqueous solution; at leastabout 41 g DBM per 100 g aqueous solution; at least about 42 g DBM per100 g aqueous solution; at least about 43 g DBM per 100 g aqueoussolution; at least about 44 g DBM per 100 g aqueous solution; at leastabout 45 g DBM per 100 g aqueous solution; at least about 46 g DBM per100 g aqueous solution; at least about 47 g DBM per 100 g aqueoussolution; at least about 48 g DBM per 100 g aqueous solution; at leastabout 49 g DBM per 100 g aqueous solution; at least about 50 g DBM per100 g aqueous solution; at least about 51 g DBM per 100 g aqueoussolution; at least about 52 g DBM per 100 g aqueous solution; at leastabout 53 g DBM per 100 g aqueous solution; at least about 54 g DBM per100 g aqueous solution; at least about 55 g DBM per 100 g aqueoussolution; at least about 56 g DBM per 100 g aqueous solution; at leastabout 57 g DBM per 100 g aqueous solution; at least about 58 g DBM per100 g aqueous solution; at least about 59 g DBM per 100 g aqueoussolution; at least about 60 g DBM per 100 g aqueous solution; at leastabout 61 g DBM per 100 g aqueous solution; at least about 62 g DBM per100 g aqueous solution; at least about 63 g DBM per 100 g aqueoussolution; at least about 64 g DBM per 100 g aqueous solution; at leastabout 65 g DBM per 100 g aqueous solution; at least about 66 g DBM per100 g aqueous solution; at least about 67 g DBM per 100 g aqueoussolution; at least about 68 g DBM per 100 g aqueous solution; at leastabout 69 g DBM per 100 g aqueous solution; at least about 70 g DBM per100 g aqueous solution; at least about 71 g DBM per 100 g aqueoussolution; at least about 72 g DBM per 100 g aqueous solution; at leastabout 73 g DBM per 100 g aqueous solution; at least about 74 g DBM per100 g aqueous solution; at least about 75 g DBM per 100 g aqueoussolution; at least about 76 g DBM per 100 g aqueous solution; at leastabout 77 g DBM per 100 g aqueous solution; at least about 78 g DBM per100 g aqueous solution; at least about 79 g DBM per 100 g aqueoussolution; at least about 80 g DBM per 100 g aqueous solution; at leastabout 81 g DBM per 100 g aqueous solution; at least about 82 g DBM per100 g aqueous solution; at least about 83 g DBM per 100 g aqueoussolution; at least about 84 g DBM per 100 g aqueous solution; at leastabout 85 g DBM per 100 g aqueous solution; at least about 86 g DBM per100 g aqueous solution; at least about 87 g DBM per 100 g aqueoussolution; at least about 88 g DBM per 100 g aqueous solution; at leastabout 89 g DBM per 100 g aqueous solution; at least about 90 g DBM per100 g aqueous solution; at least about 91 g DBM per 100 g aqueoussolution; at least about 92 g DBM per 100 g aqueous solution; at leastabout 93 g DBM per 100 g aqueous solution; at least about 94 g DBM per100 g aqueous solution; at least about 95 g DBM per 100 g aqueoussolution; at least about 96 g DBM per 100 g aqueous solution; at leastabout 97 g DBM per 100 g aqueous solution; at least about 98 g DBM per100 g aqueous solution; or at least about 99 g DBM per 100 g aqueoussolution per 100 g of aqueous solution. It is understood that recitationof the above discrete values includes a range between each recitedvalue.

As another example, the amount of DBM can comprise about 1 g DBM per 100g aqueous solution. The amount of DBM can comprise about 1 g DBM per 100g aqueous solution; about 2 g DBM per 100 g aqueous solution; about 3 gDBM per 100 g aqueous solution; about 4 g DBM per 100 g aqueoussolution; about 5 g DBM per 100 g aqueous solution; about 6 g DBM per100 g aqueous solution; about 7 g DBM per 100 g aqueous solution; about8 g DBM per 100 g aqueous solution; about 9 g DBM per 100 g aqueoussolution; about 10 g DBM per 100 g aqueous solution; about 11 g DBM per100 g aqueous solution; about 12 g DBM per 100 g aqueous solution; about13 g DBM per 100 g aqueous solution; about 14 g DBM per 100 g aqueoussolution; about 15 g DBM per 100 g aqueous solution; about 16 g DBM per100 g aqueous solution; about 17 g DBM per 100 g aqueous solution; about18 g DBM per 100 g aqueous solution; about 19 g DBM per 100 g aqueoussolution; about 20 g DBM per 100 g aqueous solution; about 21 g DBM per100 g aqueous solution; about 22 g DBM per 100 g aqueous solution; about23 g DBM per 100 g aqueous solution; about 24 g DBM per 100 g aqueoussolution; about 25 g DBM per 100 g aqueous solution; about 26 g DBM per100 g aqueous solution; about 27 g DBM per 100 g aqueous solution; about28 g DBM per 100 g aqueous solution; about 29 g DBM per 100 g aqueoussolution; about 30 g DBM per 100 g aqueous solution; about 31 g DBM per100 g aqueous solution; about 32 g DBM per 100 g aqueous solution; about33 g DBM per 100 g aqueous solution; about 34 g DBM per 100 g aqueoussolution; about 35 g DBM per 100 g aqueous solution; about 36 g DBM per100 g aqueous solution; about 37 g DBM per 100 g aqueous solution; about38 g DBM per 100 g aqueous solution; about 39 g DBM per 100 g aqueoussolution; about 40 g DBM per 100 g aqueous solution; about 41 g DBM per100 g aqueous solution; about 42 g DBM per 100 g aqueous solution; about43 g DBM per 100 g aqueous solution; about 44 g DBM per 100 g aqueoussolution; about 45 g DBM per 100 g aqueous solution; about 46 g DBM per100 g aqueous solution; about 47 g DBM per 100 g aqueous solution; about48 g DBM per 100 g aqueous solution; about 49 g DBM per 100 g aqueoussolution; about 50 g DBM per 100 g aqueous solution; about 51 g DBM per100 g aqueous solution; about 52 g DBM per 100 g aqueous solution; about53 g DBM per 100 g aqueous solution; about 54 g DBM per 100 g aqueoussolution; about 55 g DBM per 100 g aqueous solution; about 56 g DBM per100 g aqueous solution; about 57 g DBM per 100 g aqueous solution; about58 g DBM per 100 g aqueous solution; about 59 g DBM per 100 g aqueoussolution; about 60 g DBM per 100 g aqueous solution; about 61 g DBM per100 g aqueous solution; about 62 g DBM per 100 g aqueous solution; about63 g DBM per 100 g aqueous solution; about 64 g DBM per 100 g aqueoussolution; about 65 g DBM per 100 g aqueous solution; about 66 g DBM per100 g aqueous solution; about 67 g DBM per 100 g aqueous solution; about68 g DBM per 100 g aqueous solution; about 69 g DBM per 100 g aqueoussolution; about 70 g DBM per 100 g aqueous solution; about 71 g DBM per100 g aqueous solution; about 72 g DBM per 100 g aqueous solution; about73 g DBM per 100 g aqueous solution; about 74 g DBM per 100 g aqueoussolution; about 75 g DBM per 100 g aqueous solution; about 76 g DBM per100 g aqueous solution; about 77 g DBM per 100 g aqueous solution; about78 g DBM per 100 g aqueous solution; about 79 g DBM per 100 g aqueoussolution; about 80 g DBM per 100 g aqueous solution; about 81 g DBM per100 g aqueous solution; about 82 g DBM per 100 g aqueous solution; about83 g DBM per 100 g aqueous solution; about 84 g DBM per 100 g aqueoussolution; about 85 g DBM per 100 g aqueous solution; about 86 g DBM per100 g aqueous solution; about 87 g DBM per 100 g aqueous solution; about88 g DBM per 100 g aqueous solution; about 89 g DBM per 100 g aqueoussolution; about 90 g DBM per 100 g aqueous solution; about 91 g DBM per100 g aqueous solution; about 92 g DBM per 100 g aqueous solution; about93 g DBM per 100 g aqueous solution; about 94 g DBM per 100 g aqueoussolution; about 95 g DBM per 100 g aqueous solution; about 96 g DBM per100 g aqueous solution; about 97 g DBM per 100 g aqueous solution; about98 g DBM per 100 g aqueous solution; or about 99 g DBM per 100 g aqueoussolution. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

The aqueous solution can comprise the mineral coating solution asdescribed herein. The aqueous solution can comprise any biologicallycompatible aqueous solution, particularly those in which mineral coatingcomponents, growth factors, and proteins may be stable in. Examples ofsuch solutions can be, but not limited to the mineral coating solutionas described above, Tris buffer, Tris buffered saline, phosphate bufferand phosphate buffered saline. For example, the solution can be a weakacid solution where the weak acid can be, but not limited to, citricacid, lactic acid, malic acid, ascorbic acid or combinations thereof.Any weak acid known in the art can be used. The concentration of theweak acid solution can be from about 2 M to about 3 M. In a secondillustrative embodiment, the solution can be a guanidine hydrochloridesolution where the concentration of the guanidine hydrochloride solutioncan be from about 3 M to about 6 M.

The amount of time that the DBM and aqueous solution can be mixed fromabout 8 hours to about 96 hours. For example, the DBM and aqueoussolution can be mixed together from about 24 hours to about 96 hours.The DBM and aqueous solution can be mixed together with constantagitation during that time. Constant agitation can be obtained by, butnot limited to, stirring, shaking, ultrasound or any combination thereofas well as any other methods of agitating a mixture.

The mixing can be carried out at a temperature that can be conducive tocoating a scaffold and extracting growth factors and/or proteins fromthe DBM, but where growth factors and/or proteins can be stable. Thetemperature can be less than 50° C. As another example, the temperaturecan be room temperature.

After mixing for the appropriate amount of time, the resultingdemineralized bone extract can be separated from any insoluble DBMremaining in the solution. This separation can occur by any number ofprocesses such as, but not limited to, decanting, filtering, orcentrifuging. For example, the solution can be filtered to remove anysoluble DBM remaining. The size of the sieve or filter will depend onthe size of the DBM particles remaining, which can further depend on theinitial form of DBM. For example, the filter can be from about 50microns to about 300 microns. As another example, the filter can be asieve, paper, scintered glass, woven or non-woven fabric, or any othermeans of filtering that is known in the art.

The demineralized bone extract can be diluted, neutralized or the weakacid or guanidine hydrochloride removed. Methods can include, but arenot limited to, titration, dialysis, liquid-liquid extraction, hollowfiber filtration, ultrafiltration, crossflow filtration orprecipitation. For example, the aqueous solution can be neutralized to apH of from about 6.5 to about 7.5 by titration with an appropriatecounterion. Such methods are well known in the art. As another example,the weak acid or guanidine hydrochloride can be removed by dialysis,hollow fiber filtration, ultrafiltration or crossflow filtration againsta biologically compatible buffer, such as, but not limited to, Tris,TBS, phosphate, PBS or water, where the pH of the buffer can be fromabout 6.5 to about 7.5. The molecular weight cutoff of the dialysismembrane will depend on the size of the proteins and/or growth factorsdesired in the solution. The dialysis, hollow fiber filtration,ultrafiltration or crossflow filtration membrane can have, for example,a molecular weight cut off less than or equal to 12 Kd or from about 10Kd to about 12 Kd. It is well known in the art how to select themolecular weight cut off of dialysis tubing to retain the desiredmolecules within the sample.

The demineralized bone extract can include the mineral coating solutionor be mixed with the mineral coating solution as described herein. Forexample, the bone extract can be introduced during the scaffoldincubation step in the mineral coating procedure as described in Example4.

As described herein, a mineral coated scaffold can be coatedsimultaneously with the demineralized bone material or the mineralcoated scaffold can be coated with the demineralizing bone material.

The demineralized bone extract can be applied to the scaffold such thatthe demineralized bone extract infiltrates the pores and passageways ofthe scaffold. For example, the demineralized bone extract can be appliedto the scaffold under vacuum. As another example, the demineralized boneextract can be applied to the scaffold by dipping the structure into theextract and allowing it to infiltrate the pores and passageways bycapillary action. After the demineralized bone extract has been appliedto the scaffold, it can be dried onto the scaffold. The demineralizedbone extract can be dried onto the structure by lyophilization, vacuum,heating, or a combination thereof. In one oe more embodiments, theheating can be at a temperature less than 50° C.

As described herein, the method of the present invention can furthercomprise making a demineralized bone gelatin. The demineralized bonegelatin can be coated over the demineralized bone extract or mineralcoating, or it can be mixed with the extract before coating to form asingle coating or it can be mixed with the mineral coating to form asingle coating. The demineralized bone gelatin can be formed by mixingDBM with an aqueous saline solution such as, but not limited to PBS,TBS, or a sodium chloride solution, to form a suspension. The suspensioncan be treated to increased temperature and pressure such as, but notlimited to, autoclaved. In one illustrative embodiment, the solution canbe heated to a temperature of from about 85° C. to about 130° C. at apressure of at least about 15 psig. The DBM can be dissolved to producea demineralized bone gelatin. Methods for forming a demineralized bonegelatin are known in the art. The DBM can be the solids removed duringthe filtering step while forming the demineralized bone extract or itcan be fresh DBM. Alternatively, it will be appreciated that since thedemineralized bone gelatin comprises mainly collagen, collagen of anypurity can be substituted for the DBM.

The demineralized bone gelatin can be coated over the drieddemineralized bone extract coating on the scaffold such that the gelatincoats the pores and passageways. Alternatively, the demineralized bonegelatin can be mixed with the demineralized bone extract prior to theextract being coated onto the scaffold to form a single coating. Thescaffold can be pre-coated with the mineral coating or the mineralcoating solution can be incorporated into the DBM coating. The coatingcomprising the demineralized bone extract and gelatin can then beapplied to the scaffold such that the pores and passageways are coated.It will be appreciated that the demineralized bone gelatin can be lessviscous at higher temperatures, making it easier to apply to thescaffold. The demineralized bone gelatin can be applied to the scaffoldin a less viscous form such as a solution. The demineralized bonegelatin can be allowed to gel before any other steps are performed. Thedemineralized bone gelatin coating can be maintained at a temperaturelow enough as to not to inactivate any growth factors and/or proteins ofthe demineralized bone extract coating.

Once applied, the demineralized bone gelatin, either alone or mixed withthe demineralized bone extract or mineral coating, can be dried onto thescaffold. The scaffold can be pre-coated with the mineral coating. Thedemineralized bone gelatin can be dried onto the structure bylyophilization, vacuum, heating, or a combination thereof. In one oemore embodiments, the heating can be at a temperature not greater than50° C.

As described herein, DBM can be integrated into the mineral coating orinto a scaffold. For example, DBM can be integrated into the mineralcoating or scaffold by % weight (w/w) or by % volume (v/v). A mineralcoating or scaffold can comprise at least about 1% (w/w) DBM. Forexample, a mineral coating or scaffold can comprise at least about 1%(w/w) DBM; at least about 2% (w/w) DBM; at least about 3% (w/w) DBM; atleast about 4% (w/w) DBM; at least about 5% (w/w) DBM; at least about 6%(w/w) DBM; at least about 7% (w/w) DBM; at least about 8% (w/w) DBM; atleast about 9% (w/w) DBM; at least about 10% (w/w) DBM; at least about11% (w/w) DBM; at least about 12% (w/w) DBM; at least about 13% (w/w)DBM; at least about 14% (w/w) DBM; at least about 15% (w/w) DBM; atleast about 16% (w/w) DBM; at least about 17% (w/w) DBM; at least about18% (w/w) DBM; at least about 19% (w/w) DBM; at least about 20% (w/w)DBM; at least about 21% (w/w) DBM; at least about 22% (w/w) DBM; atleast about 23% (w/w) DBM; at least about 24% (w/w) DBM; at least about25% (w/w) DBM; at least about 26% (w/w) DBM; at least about 27% (w/w)DBM; at least about 28% (w/w) DBM; at least about 29% (w/w) DBM; atleast about 30% (w/w) DBM; at least about 31% (w/w) DBM; at least about32% (w/w) DBM; at least about 33% (w/w) DBM; at least about 34% (w/w)DBM; at least about 35% (w/w) DBM; at least about 36% (w/w) DBM; atleast about 37% (w/w) DBM; at least about 38% (w/w) DBM; at least about39% (w/w) DBM; at least about 40% (w/w) DBM; at least about 41% (w/w)DBM; at least about 42% (w/w) DBM; at least about 43% (w/w) DBM; atleast about 44% (w/w) DBM; at least about 45% (w/w) DBM; at least about46% (w/w) DBM; at least about 47% (w/w) DBM; at least about 48% (w/w)DBM; at least about 49% (w/w) DBM; at least about 50% (w/w) DBM; atleast about 51% (w/w) DBM; at least about 52% (w/w) DBM; at least about53% (w/w) DBM; at least about 54% (w/w) DBM; at least about 55% (w/w)DBM; at least about 56% (w/w) DBM; at least about 57% (w/w) DBM; atleast about 58% (w/w) DBM; at least about 59% (w/w) DBM; at least about60% (w/w) DBM; at least about 61% (w/w) DBM; at least about 62% (w/w)DBM; at least about 63% (w/w) DBM; at least about 64% (w/w) DBM; atleast about 65% (w/w) DBM; at least about 66% (w/w) DBM; at least about67% (w/w) DBM; at least about 68% (w/w) DBM; at least about 69% (w/w)DBM; at least about 70% (w/w) DBM; at least about 71% (w/w) DBM; atleast about 72% (w/w) DBM; at least about 73% (w/w) DBM; at least about74% (w/w) DBM; at least about 75% (w/w) DBM; at least about 76% (w/w)DBM; at least about 77% (w/w) DBM; at least about 78% (w/w) DBM; atleast about 79% (w/w) DBM; at least about 80% (w/w) DBM; at least about81% (w/w) DBM; at least about 82% (w/w) DBM; at least about 83% (w/w)DBM; at least about 84% (w/w) DBM; at least about 85% (w/w) DBM; atleast about 86% (w/w) DBM; at least about 87% (w/w) DBM; at least about88% (w/w) DBM; at least about 89% (w/w) DBM; at least about 90% (w/w)DBM; at least about 91% (w/w) DBM; at least about 92% (w/w) DBM; atleast about 93% (w/w) DBM; at least about 94% (w/w) DBM; at least about95% (w/w) DBM; at least about 96% (w/w) DBM; at least about 97% (w/w)DBM; at least about 98% (w/w) DBM; or at least about 99% (w/w) DBM. Itis understood that recitation of the above discrete values includes arange between each recited value.

As another example, a mineral coating or scaffold can comprise about 1%(w/w) DBM; about 2% (w/w) DBM; about 3% (w/w) DBM; about 4% (w/w) DBM;about 5% (w/w) DBM; about 6% (w/w) DBM; about 7% (w/w) DBM; about 8%(w/w) DBM; about 9% (w/w) DBM; about 10% (w/w) DBM; about 11% (w/w) DBM;about 12% (w/w) DBM; about 13% (w/w) DBM; about 14% (w/w) DBM; about 15%(w/w) DBM; about 16% (w/w) DBM; about 17% (w/w) DBM; about 18% (w/w)DBM; about 19% (w/w) DBM; about 20% (w/w) DBM; about 21% (w/w) DBM;about 22% (w/w) DBM; about 23% (w/w) DBM; about 24% (w/w) DBM; about 25%(w/w) DBM; about 26% (w/w) DBM; about 27% (w/w) DBM; about 28% (w/w)DBM; about 29% (w/w) DBM; about 30% (w/w) DBM; about 31% (w/w) DBM;about 32% (w/w) DBM; about 33% (w/w) DBM; about 34% (w/w) DBM; about 35%(w/w) DBM; about 36% (w/w) DBM; about 37% (w/w) DBM; about 38% (w/w)DBM; about 39% (w/w) DBM; about 40% (w/w) DBM; about 41% (w/w) DBM;about 42% (w/w) DBM; about 43% (w/w) DBM; about 44% (w/w) DBM; about 45%(w/w) DBM; about 46% (w/w) DBM; about 47% (w/w) DBM; about 48% (w/w)DBM; about 49% (w/w) DBM; about 50% (w/w) DBM; about 51% (w/w) DBM;about 52% (w/w) DBM; about 53% (w/w) DBM; about 54% (w/w) DBM; about 55%(w/w) DBM; about 56% (w/w) DBM; about 57% (w/w) DBM; about 58% (w/w)DBM; about 59% (w/w) DBM; about 60% (w/w) DBM; about 61% (w/w) DBM;about 62% (w/w) DBM; about 63% (w/w) DBM; about 64% (w/w) DBM; about 65%(w/w) DBM; about 66% (w/w) DBM; about 67% (w/w) DBM; about 68% (w/w)DBM; about 69% (w/w) DBM; about 70% (w/w) DBM; about 71% (w/w) DBM;about 72% (w/w) DBM; about 73% (w/w) DBM; about 74% (w/w) DBM; about 75%(w/w) DBM; about 76% (w/w) DBM; about 77% (w/w) DBM; about 78% (w/w)DBM; about 79% (w/w) DBM; about 80% (w/w) DBM; about 81% (w/w) DBM;about 82% (w/w) DBM; about 83% (w/w) DBM; about 84% (w/w) DBM; about 85%(w/w) DBM; about 86% (w/w) DBM; about 87% (w/w) DBM; about 88% (w/w)DBM; about 89% (w/w) DBM; about 90% (w/w) DBM; about 91% (w/w) DBM;about 92% (w/w) DBM; about 93% (w/w) DBM; about 94% (w/w) DBM; about 95%(w/w) DBM; about 96% (w/w) DBM; about 97% (w/w) DBM; about 98% (w/w)DBM; or about 99% (w/w) DBM. It is understood that recitation of theabove discrete values includes a range between each recited value.

A mineral coating or scaffold can comprise at least about 1% (v/v) DBM.As another example, a mineral coating or scaffold can comprise at leastabout 1% (v/v) DBM; at least about 2% (v/v) DBM; at least about 3% (v/v)DBM; at least about 4% (v/v) DBM; at least about 5% (v/v) DBM; at leastabout 6% (v/v) DBM; at least about 7% (v/v) DBM; at least about 8% (v/v)DBM; at least about 9% (v/v) DBM; at least about 10% (v/v) DBM; at leastabout 11% (v/v) DBM; at least about 12% (v/v) DBM; at least about 13%(v/v) DBM; at least about 14% (v/v) DBM; at least about 15% (v/v) DBM;at least about 16% (v/v) DBM; at least about 17% (v/v) DBM; at leastabout 18% (v/v) DBM; at least about 19% (v/v) DBM; at least about 20%(v/v) DBM; at least about 21% (v/v) DBM; at least about 22% (v/v) DBM;at least about 23% (v/v) DBM; at least about 24% (v/v) DBM; at leastabout 25% (v/v) DBM; at least about 26% (v/v) DBM; at least about 27%(v/v) DBM; at least about 28% (v/v) DBM; at least about 29% (v/v) DBM;at least about 30% (v/v) DBM; at least about 31% (v/v) DBM; at leastabout 32% (v/v) DBM; at least about 33% (v/v) DBM; at least about 34%(v/v) DBM; at least about 35% (v/v) DBM; at least about 36% (v/v) DBM;at least about 37% (v/v) DBM; at least about 38% (v/v) DBM; at leastabout 39% (v/v) DBM; at least about 40% (v/v) DBM; at least about 41%(v/v) DBM; at least about 42% (v/v) DBM; at least about 43% (v/v) DBM;at least about 44% (v/v) DBM; at least about 45% (v/v) DBM; at leastabout 46% (v/v) DBM; at least about 47% (v/v) DBM; at least about 48%(v/v) DBM; at least about 49% (v/v) DBM; at least about 50% (v/v) DBM;at least about 51% (v/v) DBM; at least about 52% (v/v) DBM; at leastabout 53% (v/v) DBM; at least about 54% (v/v) DBM; at least about 55%(v/v) DBM; at least about 56% (v/v) DBM; at least about 57% (v/v) DBM;at least about 58% (v/v) DBM; at least about 59% (v/v) DBM; at leastabout 60% (v/v) DBM; at least about 61% (v/v) DBM; at least about 62%(v/v) DBM; at least about 63% (v/v) DBM; at least about 64% (v/v) DBM;at least about 65% (v/v) DBM; at least about 66% (v/v) DBM; at leastabout 67% (v/v) DBM; at least about 68% (v/v) DBM; at least about 69%(v/v) DBM; at least about 70% (v/v) DBM; at least about 71% (v/v) DBM;at least about 72% (v/v) DBM; at least about 73% (v/v) DBM; at leastabout 74% (v/v) DBM; at least about 75% (v/v) DBM; at least about 76%(v/v) DBM; at least about 77% (v/v) DBM; at least about 78% (v/v) DBM;at least about 79% (v/v) DBM; at least about 80% (v/v) DBM; at leastabout 81% (v/v) DBM; at least about 82% (v/v) DBM; at least about 83%(v/v) DBM; at least about 84% (v/v) DBM; at least about 85% (v/v) DBM;at least about 86% (v/v) DBM; at least about 87% (v/v) DBM; at leastabout 88% (v/v) DBM; at least about 89% (v/v) DBM; at least about 90%(v/v) DBM; at least about 91% (v/v) DBM; at least about 92% (v/v) DBM;at least about 93% (v/v) DBM; at least about 94% (v/v) DBM; at leastabout 95% (v/v) DBM; at least about 96% (v/v) DBM; at least about 97%(v/v) DBM; at least about 98% (v/v) DBM; or at least about 99% (v/v)DBM. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

As another example, a mineral coating or scaffold can comprise about 1%(v/v) DBM; about 2% (v/v) DBM; about 3% (v/v) DBM; about 4% (v/v) DBM;about 5% (v/v) DBM; about 6% (v/v) DBM; about 7% (v/v) DBM; about 8%(v/v) DBM; about 9% (v/v) DBM; about 10% (v/v) DBM; about 11% (v/v) DBM;about 12% (v/v) DBM; about 13% (v/v) DBM; about 14% (v/v) DBM; about 15%(v/v) DBM; about 16% (v/v) DBM; about 17% (v/v) DBM; about 18% (v/v)DBM; about 19% (v/v) DBM; about 20% (v/v) DBM; about 21% (v/v) DBM;about 22% (v/v) DBM; about 23% (v/v) DBM; about 24% (v/v) DBM; about 25%(v/v) DBM; about 26% (v/v) DBM; about 27% (v/v) DBM; about 28% (v/v)DBM; about 29% (v/v) DBM; about 30% (v/v) DBM; about 31% (v/v) DBM;about 32% (v/v) DBM; about 33% (v/v) DBM; about 34% (v/v) DBM; about 35%(v/v) DBM; about 36% (v/v) DBM; about 37% (v/v) DBM; about 38% (v/v)DBM; about 39% (v/v) DBM; about 40% (v/v) DBM; about 41% (v/v) DBM;about 42% (v/v) DBM; about 43% (v/v) DBM; about 44% (v/v) DBM; about 45%(v/v) DBM; about 46% (v/v) DBM; about 47% (v/v) DBM; about 48% (v/v)DBM; about 49% (v/v) DBM; about 50% (v/v) DBM; about 51% (v/v) DBM;about 52% (v/v) DBM; about 53% (v/v) DBM; about 54% (v/v) DBM; about 55%(v/v) DBM; about 56% (v/v) DBM; about 57% (v/v) DBM; about 58% (v/v)DBM; about 59% (v/v) DBM; about 60% (v/v) DBM; about 61% (v/v) DBM;about 62% (v/v) DBM; about 63% (v/v) DBM; about 64% (v/v) DBM; about 65%(v/v) DBM; about 66% (v/v) DBM; about 67% (v/v) DBM; about 68% (v/v)DBM; about 69% (v/v) DBM; about 70% (v/v) DBM; about 71% (v/v) DBM;about 72% (v/v) DBM; about 73% (v/v) DBM; about 74% (v/v) DBM; about 75%(v/v) DBM; about 76% (v/v) DBM; about 77% (v/v) DBM; about 78% (v/v)DBM; about 78% (v/v) DBM; about 78% (v/v) DBM; about 79% (v/v) DBM;about 80% (v/v) DBM; about 81% (v/v) DBM; about 82% (v/v) DBM; about 83%(v/v) DBM; about 84% (v/v) DBM; about 85% (v/v) DBM; about 86% (v/v)DBM; about 87% (v/v) DBM; about 88% (v/v) DBM; about 89% (v/v) DBM;about 90% (v/v) DBM; about 91% (v/v) DBM; about 92% (v/v) DBM; about 93%(v/v) DBM; about 94% (v/v) DBM; about 95% (v/v) DBM; about 96% (v/v)DBM; about 97% (v/v) DBM; about 98% (v/v) DBM; or about 99% (v/v) DBM.It is understood that recitation of the above discrete values includes arange between each recited value.

As described herein, an auxiliary component can be an antimicrobialagent. For example, an antimicrobial agent can be silver particles. Asanother example, silver particles can be silver microparticles or silvernanoparticles. Silver particles integrated with the scaffold arepresently thought to provide biostatic, anti-infection properties to thescaffold.

As described herein, silver particles can be integrated onto the mineralcoating or scaffold. For example, the silver particles can be integratedinto the scaffold or coating before, during, or after DBM integration.

As described herein, silver particles can be incorporated onto thesurface of a mineral coated scaffold. For example, the silver particlescan be incorporated into or onto the mineral coating by any method knownin the art. As another example, the incorporation of silver particlescan be as described in Lee et al. 2013 Mat. Views 25, 1173-1179; WO2014/110284; U.S. Pat. No. 8,673,018; US 2013/0142885; Ciobanu (2014);Jadalannagari (2014); US 2009/0198344.

As described herein, mineral coated scaffolds can be incubated in citricacid solution and silver nitrate solution to produce silvernanoparticles and microparticles on the mineral coating.

As described herein, the mineral coated scaffold can be incubated incitric acid solution. For example, the citric acid solution can be from0.1 to 100 mM citric acid solution. As another example, theconcentration of citric acid solution can be at least about 0.1 mMcitric acid. As another example, the concentration of citric acidsolution can be at least about 0.1 mM citric acid; at least about 0.2 mMcitric acid; at least about 0.3 mM citric acid; at least about 0.4 mMcitric acid; at least about 0.5 mM citric acid; at least about 0.6 mMcitric acid; at least about 0.7 mM citric acid; at least about 0.8 mMcitric acid; at least about 0.9 mM citric acid; at least about 1 mMcitric acid; at least about 1.5 mM citric acid; at least about 2 mMcitric acid; at least about 2.5 mM citric acid; at least about 3.0 mMcitric acid; at least about 3.5 mM citric acid; at least about 4.0 mMcitric acid; at least about 4.5 mM citric acid; at least about 5.0 mMcitric acid; at least about 5.5 mM citric acid; at least about 6.0 mMcitric acid; at least about 6.5 mM citric acid; at least about 7.0 mMcitric acid; at least about 7.5 mM citric acid; at least about 8.0 mMcitric acid; at least about 8.5 mM citric acid; at least about 9.0 mMcitric acid; at least about 10 mM citric acid; at least about 15 mMcitric acid; at least about 20 mM citric acid; at least about 25 mMcitric acid; at least about 30 mM citric acid; at least about 35 mMcitric acid; at least about 40 mM citric acid; at least about 45 mMcitric acid; at least about 50 mM citric acid; at least about 55 mMcitric acid; at least about 60 mM citric acid; at least about 65 mMcitric acid; at least about 70 mM citric acid; at least about 75 mMcitric acid; at least about 80 mM citric acid; at least about 85 mMcitric acid; at least about 90 mM citric acid; at least about 95 mMcitric acid; at least about; or 100 mM citric acid. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

As described herein, the concentration of citric acid solution can beabout 0.1 mM citric acid; at least about 0.2 mM citric acid; about 0.3mM citric acid; about 0.4 mM citric acid; about 0.5 mM citric acid;about 0.6 mM citric acid; about 0.7 mM citric acid; about 0.8 mM citricacid; about 0.9 mM citric acid; about 1 mM citric acid; about 1.5 mMcitric acid; about 2 mM citric acid; about 2.5 mM citric acid; about 3.0mM citric acid; about 3.5 mM citric acid; about 4.0 mM citric acid;about 4.5 mM citric acid; about 5.0 mM citric acid; about 5.5 mM citricacid; about 6.0 mM citric acid; about 6.5 mM citric acid; about 7.0 mMcitric acid; about 7.5 mM citric acid; about 8.0 mM citric acid; about8.5 mM citric acid; about 9.0 mM citric acid; about 10 mM citric acid;about 15 mM citric acid; about 20 mM citric acid; about 25 mM citricacid; about 30 mM citric acid; about 35 mM citric acid; about 40 mMcitric acid; about 45 mM citric acid; about 50 mM citric acid; about 55mM citric acid; about 60 mM citric acid; about 65 mM citric acid; about70 mM citric acid; about 75 mM citric acid; about 80 mM citric acid;about 85 mM citric acid; about 90 mM citric acid; about 95 mM citricacid; about; or 100 mM citric acid. It is understood that recitation ofthe above discrete values includes a range between each recited value.

As described herein, the mineral coated scaffold can be incubated incitric acid solution for 0.1 hours to 40 hours. For example, the mineralcoated scaffold can be incubated for 0.5 hours to 4 hours. It isunderstood that recitation of the above ranges includes discrete valueswithin each recited range.

As described herein, the mineral coated scaffold can be incubated incitric acid solution for at least about 0.1 hours. For example, themineral coated scaffold can be incubated in citric acid solution for atleast about 0.1 hours; at least about 0.2 hours; at least about 0.3hours; at least about 0.4 hours; at least about 0.5 hours; at leastabout 0.6 hours; at least about 0.7 hours; at least about 0.8 hours; atleast about 0.9 hours; at least about 1 hours; at least about 1.1 hours;at least about 1.2 hours; at least about 1.3 hours; at least about 1.4hours; at least about 1.5 hours; at least about 1.6 hours; at leastabout 1.7 hours; at least about 1.8 hours; at least about 1.9 hours; atleast about 2 hours; at least about 2.1 hours; at least about 2.3 hours;at least about 2.4 hours; at least about 2.5 hours; at least about 2.6hours; at least about 2.7 hours; at least about 2.8 hours; at leastabout 2.9 hours; at least about 3.0 hours; at least about 3.1 hours; atleast about 3.2 hours; at least about 3.3 hours; at least about 3.4hours; at least about 3.5 hours; at least about 3.6 hours; at leastabout 3.7 hours; at least about 3.8 hours; at least about 3.9 hours; atleast about 4.0 hours; at least about 5 hours; at least about 5.5 hours;at least about 6.0 hours; at least about 6.5 hours; at least about 7.5hours; at least about 8.0 hours; at least about 8.5 hours; at leastabout 9.0 hours; at least about 9.5 hours; at least about 10 hours; atleast about 11 hours; at least about 12 hours; at least about 13 hours;at least about 14 hours; at least about 15 hours; at least about 16hours; at least about 17 hours; at least about 18 hours; at least about19 hours; at least about 20 hours; at least about 21 hours; at leastabout 22 hours; at least about 23 hours; at least about 24 hours; atleast about 25 hours; at least about 26 hours; at least about 27 hours;at least about 28 hours; at least about 29 hours; at least about 30hours; at least about 31 hours; at least about 32 hours; at least about33 hours; at least about 34 hours; at least about 35 hours; at leastabout 36 hours; at least about 37 hours; at least about 38 hours; atleast about 39 hours; or at least about 40 hours. It is understood thatrecitation of the above discrete values includes a range between eachrecited value.

As another example, the mineral coated scaffold can be incubated incitric acid solution for about 0.1 hours; about 0.2 hours; about 0.3hours; about 0.4 hours; about 0.5 hours; about 0.6 hours; about 0.7hours; about 0.8 hours; about 0.9 hours; about 1 hours; about 1.1 hours;about 1.2 hours; about 1.3 hours; about 1.4 hours; about 1.5 hours;about 1.6 hours; about 1.7 hours; about 1.8 hours; about 1.9 hours;about 2.0 hours; about 2.1 hours; about 2.3 hours; about 2.4 hours;about 2.5 hours; about 2.6 hours; about 2.7 hours; about 2.8 hours;about 2.9 hours; about 3.0 hours; about 3.1 hours; about 3.2 hours;about 3.3 hours; about 3.4 hours; about 3.5 hours; about 3.6 hours;about 3.7 hours; about 3.8 hours; about 3.9 hours; about 4.0 hours;about 5 hours; about 5.5 hours; about 6.0 hours; about 6.5 hours; about7.5 hours; about 8.0 hours; about 8.5 hours; about 9.0 hours; about 9.5hours; about 10 hours; about 11 hours; about 12 hours; about 13 hours;about 14 hours; about 15 hours; about 16 hours; about 17 hours; about 18hours; about 19 hours; about 20 hours; about 21 hours; about 22 hours;about 23 hours; about 24 hours; about 25 hours; about 26 hours; about 27hours; about 28 hours; about 29 hours; about 30 hours; about 31 hours;about 32 hours; about 33 hours; about 34 hours; about 35 hours; about 36hours; about 37 hours; about 38 hours; about 39 hours; or about 40hours. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

The citric acid-treated mineral-coated scaffolds can then be transferredto a silver nitrate solution to grow silver particles. Silver particlescan cover the entire coated scaffold. The size of silver particles canbe increased with longer silver nitrate incubation time and highersilver nitrate concentrations, whereas citric acid incubation time andconcentration have not been shown to influence the silver particle size.

As described herein, silver particles can be synthesized on both citricacid-treated and non-citric-acid-treated mineral-coated scaffolds,because the size and morphology of the silver particles between thesetwo groups can be the same. Silver carbonate and silver phosphateparticles can be created locally on a mineral coated scaffold by thereaction of silver ions with carbonate or phosphate ions dissolved fromCaP in a mineral coating.

As described herein, the time course release of silver which wasprepared from different incubating conditions can be measured. Thesilver release from mineral coatings can continue for time periodsranging from 3 days to over 30 days, and the total quantity of releasedsilver species can range from 0.7 μg to 75.4 μg per cm² of samplesurface. The silver release can occur with nearly linear releasekinetics in groups treated with citric acid, while the groups nottreated with citric acid can show an initial burst release during thefirst two days. These release kinetics can be dictated by the differentdissolution rates of CaP coatings in presence of adsorbed citric acidmolecules. Burst release can pose a practical problem in the applicationof drug delivery systems, because it can occur in an unpredictablemanner and may cause negative side effects due to overdose of thereleased drug. Citric acid treatment can help avoid burst release ofsilver species, and thereby prevent complications that may be related tosilver overdose. The 4-hour incubation in citric acid solution can leadto more rapid release kinetics compared to 0.5 and 1 hour incubations.The silver release kinetics was not shown to be influenced by the citricacid concentration. This trend suggests that the amount of releasedsilver can be dictated by the adsorbed citric acid and thus dissolutionof mineral coatings. Increase of incubation time (e.g., 0.5 to 4 hr) insilver nitrate solution can result in a larger amount of silverreleased.

Similarly, higher concentrations of silver nitrate solution duringgrowth of silver particles can lead to larger quantities of silverreleased over longer release periods. Taken together, these results canindicate that the dosage and timeframe of silver release could bereadily controlled by varying the conditions during growth of silversalt particles on the mineral coated scaffold.

The antibacterial activity of released silver can be evaluated against abacterial culture. For example, against Staphylococcus and gram-negativeEscherichia coli. Media from silver-releasing CaP coatings can be addedto bacterial suspensions in their exponential growth phase, andbacterial growth can be monitored by measuring optical density at 600nm.

Silver released into the media can have antibacterial activity that issimilar against S. aureus and E. coli. Silver released at a later timepoint can remain antimicrobially active. Because previous studiesreported that citric acid can be effective to treat chronic woundinfection by preventing colony formation of microorganism, the citricacid can also be beneficial to the antibacterial properties of thesilver nanoparticle-incorporated mineral-coated scaffold.

As described herein, silver particles can cover the surface of themineral coating or scaffold with at least about 1% coverage. Forexample, the silver particles can cover the surface of the mineralcoating or scaffold with at least about 1% coverage; at least about 2%coverage; at least about 3% coverage; at least about 4% coverage; atleast about 5% coverage; at least about 6% coverage; at least about 7%coverage; at least about 8% coverage; at least about 9% coverage; atleast about 10% coverage; at least about 11% coverage; at least about12% coverage; at least about 13% coverage; at least about 14% coverage;at least about 15% coverage; at least about 16% coverage; at least about17% coverage; at least about 18% coverage; at least about 19% coverage;at least about 20% coverage; at least about 21% coverage; at least about22% coverage; at least about 23% coverage; at least about 24% coverage;at least about 25% coverage; at least about 26% coverage; at least about27% coverage; at least about 28% coverage; at least about 29% coverage;at least about 30% coverage; at least about 31% coverage; at least about32% coverage; at least about 33% coverage; at least about 34% coverage;at least about 35% coverage; at least about 36% coverage; at least about37% coverage; at least about 38% coverage; at least about 39% coverage;at least about 40% coverage; at least about 41% coverage; at least about42% coverage; at least about 43% coverage; at least about 44% coverage;at least about 45% coverage; at least about 46% coverage; at least about47% coverage; at least about 48% coverage; at least about 49% coverage;at least about 50% coverage; at least about 51% coverage; at least about52% coverage; at least about 53% coverage; at least about 54% coverage;at least about 55% coverage; at least about 56% coverage; at least about57% coverage; at least about 58% coverage; at least about 59% coverage;at least about 60% coverage; at least about 61% coverage; at least about62% coverage; at least about 63% coverage; at least about 64% coverage;at least about 65% coverage; at least about 66% coverage; at least about67% coverage; at least about 68% coverage; at least about 69% coverage;at least about 70% coverage; at least about 71% coverage; at least about72% coverage; at least about 73% coverage; at least about 74% coverage;at least about 75% coverage; at least about 76% coverage; at least about77% coverage; at least about 78% coverage; at least about 79% coverage;at least about 80% coverage; at least about 81% coverage; at least about82% coverage; at least about 83% coverage; at least about 84% coverage;at least about 85% coverage; at least about 86% coverage; at least about87% coverage; at least about 88% coverage; at least about 89% coverage;at least about 90% coverage; at least about 91% coverage; at least about92% coverage; at least about 93% coverage; at least about 94% coverage;at least about 95% coverage; at least about 96% coverage; at least about97% coverage; at least about 98% coverage; or at least about 99%coverage. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

As described herein, the silver particles can cover the surface of themineral coating or scaffold with at least about 1% coverage; about 2%coverage; about 3% coverage; about 4% coverage; about 5% coverage; about6% coverage; about 7% coverage; about 8% coverage; about 9% coverage;about 10% coverage; about 11% coverage; about 12% coverage; about 13%coverage; about 14% coverage; about 15% coverage; about 16% coverage;about 17% coverage; about 18% coverage; about 19% coverage; about 20%coverage; about 21% coverage; about 22% coverage; about 23% coverage;about 24% coverage; about 25% coverage; about 26% coverage; about 27%coverage; about 28% coverage; about 29% coverage; about 30% coverage;about 31% coverage; about 32% coverage; about 33% coverage; about 34%coverage; about 35% coverage; about 36% coverage; about 37% coverage;about 38% coverage; about 39% coverage; about 40% coverage; about 41coverage; about 42% coverage; about 43% coverage; about 44% coverage;about 45% coverage; about 46% coverage; about 47% coverage; about 48%coverage; about 49% coverage; about 50% coverage; about 51% coverage;about 52% coverage; about 53% coverage; about 54% coverage; about 55%coverage; about 56% coverage; about 57% coverage; about 58% coverage;about 59% coverage; about 60% coverage; about 61% coverage; about 62%coverage; about 63% coverage; about 64% coverage; about 65% coverage;about 66% coverage; about 67% coverage; about 68% coverage; about 69%coverage; about 70% coverage; about 71% coverage; about 72% coverage;about 73% coverage; about 74% coverage; about 75% coverage; about 76%coverage; about 77% coverage; about 78% coverage; about 79% coverage;about 80% coverage; about 81% coverage; about 82% coverage; about 83%coverage; about 84% coverage; about 85% coverage; about 86% coverage;about 87% coverage; about 88% coverage; about 89% coverage; about 90%coverage; about 91% coverage; about 92% coverage; about 93% coverage;about 94% coverage; about 95% coverage; about 96% coverage; about 97%coverage; about 98% coverage; or about 99% coverage. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

Although the embodiments described herein describe single layers ofcoatings, more than one coating or application of the mineral coating,DBM, or silver particles may be applied.

Buffers

The buffer, as described herein, can be used in the modified simulatedbody fluid. The buffer, as described herein, can be any conventionalbuffer. A buffer can be as described in U.S. application Ser. Nos.13/407,441; 13/879,178; and 13/036,470 and are incorporated byreference.

For example, the buffer can be a saline buffer. As another example, thebuffer can be Tris. Tris can be tris-buffered saline (TBS). Tris can beTris HCl. As another example, the buffer can be PBS. PBS can be DPBS. Asanother example a buffer can be a mixture including citric acid,monopotassium phosphate, boric acid, or diethyl barbituric acid. Asanother example a buffer can be TAPS, Bicine, Tricine, TAPSO, HEES, TES,MOPS, PIPES, Cacodylate, SSC, MES, or succinic acid.

The buffer, as described herein, can be at any conventionalconcentration. For example, the concentration of buffer in the simulatedbody fluid can be at least about 1 mM buffer. As another example, theconcentration of buffer in the simulated body fluid can be at leastabout 2 mM buffer; at least about 3 mM buffer; at least about 4 mMbuffer; at least about 5 mM buffer; at least about 6 mM buffer; at leastabout 7 mM buffer; at least about 8 mM buffer; at least about 9 mMbuffer; at least about 10 mM buffer; at least about 11 mM buffer; atleast about 12 mM buffer; at least about 13 mM buffer; at least about 14mM buffer; at least about 15 mM buffer; at least about 16 mM buffer; atleast about 17 mM buffer; at least about 18 mM buffer; at least about 19mM buffer; at least about 20 mM buffer; at least about 21 mM buffer; atleast about 22 mM buffer; at least about 23 mM buffer; at least about 24mM buffer; at least about 25 mM buffer; at least about 26 mM buffer; atleast about 27 mM buffer; at least about 28 mM buffer; at least about 29mM buffer; at least about 30 mM buffer; at least about 31 mM buffer; atleast about 32 mM buffer; at least about 33 mM buffer; at least about 34mM buffer; at least about 35 mM buffer; at least about 36 mM buffer; atleast about 37 mM buffer; at least about 38 mM buffer; at least about 39mM buffer; at least about 40 mM buffer; at least about 50 mM buffer; atleast about 60 mM buffer; at least about 70 mM buffer; at least about 80mM buffer; at least about 90 mM buffer; at least about 100 mM buffer; atleast about 110 mM buffer; at least about 120 mM buffer; at least about130 mM buffer; at least about 140 mM buffer; at least about 150 mMbuffer; at least about 160 mM buffer; at least about 170 mM buffer; atleast about 180 mM buffer; at least about 190 mM buffer; at least about120 mM buffer; at least about 130 mM buffer; at least about 140 mMbuffer; at least about 150 mM buffer; at least about 160 mM buffer; atleast about 170 mM buffer; at least about 180 mM buffer; at least about190 mM buffer; or at least about 200 mM buffer. It is understood thatrecitation of the above discrete values includes a range between eachrecited value.

As another example, the concentration of buffer in the simulated bodyfluid can be about 1 mM buffer; about 2 mM buffer; about 3 mM buffer;about 4 mM buffer; about 5 mM buffer; about 6 mM buffer; about 7 mMbuffer; about 8 mM buffer; about 9 mM buffer; about 10 mM buffer; about11 mM buffer; about 12 mM buffer; about 13 mM buffer; about 14 mMbuffer; about 15 mM buffer; about 16 mM buffer; about 17 mM buffer;about 18 mM buffer; about 19 mM buffer; about 20 mM buffer; about 21 mMbuffer; about 22 mM buffer; about 23 mM buffer; about 24 mM buffer;about 25 mM buffer; about 26 mM buffer; about 27 mM buffer; about 28 mMbuffer; about 29 mM buffer; about 30 mM buffer; about 31 mM buffer;about 32 mM buffer; about 33 mM buffer; about 34 mM buffer; about 35 mMbuffer; about 36 mM buffer; about 37 mM buffer; about 38 mM buffer;about 39 mM buffer; about 40 mM buffer; about 50 mM buffer; about 60 mMbuffer; about 70 mM buffer; about 80 mM buffer; about 90 mM buffer;about 100 mM buffer; about 110 mM buffer; about 120 mM buffer; about 130mM buffer; about 140 mM buffer; about 150 mM buffer; about 160 mMbuffer; about 170 mM buffer; about 180 mM buffer; about 190 mM buffer;about 120 mM buffer; about 130 mM buffer; about 140 mM buffer; about 150mM buffer; about 160 mM buffer; about 170 mM buffer; about 180 mMbuffer; about 190 mM buffer; or about 200 mM buffer. It is understoodthat recitation of the above discrete values includes a range betweeneach recited value.

A buffer can be held at any pH conventional in the art. For example, abuffer can have a pH in the range of 2 to 11. As another example, abuffer can have a pH value of at least about 2. As another example, abuffer can have a pH value of at least about 2; a pH value of at leastabout 2.5; a pH value of at least about 3; a pH value of at least about3.5; a pH value of at least about 4; a pH value of at least about 4.5; apH value of at least about 5; a pH value of at least about 5.5; a pHvalue of at least about 6; a pH value of at least about 6.1; a pH valueof at least about 6.2; a pH value of at least about 6.3; a pH value ofat least about 6.4; a pH value of at least about 6.5; a pH value of atleast about 6.6; a pH value of at least about 6.7; a pH value of atleast about 6.8; a pH value of at least about 6.9; a pH value of atleast about 7; a pH value of at least about 7.1; a pH value of at leastabout 7.2; a pH value of at least about 7.3; a pH value of at leastabout 7.4; a pH value of at least about 7.5; a pH value of at leastabout 7.6; a pH value of at least about 7.7; a pH value of at leastabout 7.8; a pH value of at least about 7.9; a pH value of at leastabout 8; a pH value of at least about 8.5; a pH value of at least about9; a pH value of at least about 9.5; a pH value of at least about 10; apH value of at least about 11. It is understood that recitation of theabove discrete values includes a range between each recited value.

As another example, a buffer can have a pH value of about 2; a pH valueof about 2.5; a pH value of about 3; a pH value of about 3.5; a pH valueof about 4; a pH value of about 4.5; a pH value of about 5; a pH valueof about 5.5; a pH value of about 6; a pH value of about 6.1; a pH valueof about 6.2; a pH value of about 6.3; a pH value of about 6.4; a pHvalue of about 6.5; a pH value of about 6.6; a pH value of about 6.7; apH value of about 6.8; a pH value of about 6.9; a pH value of about 7; apH value of about 7.1; a pH value of about 7.2; a pH value of about 7.3;a pH value of about 7.4; a pH value of about 7.5; a pH value of about7.6; a pH value of about 7.7; a pH value of about 7.8; a pH value ofabout 7.9; a pH value of about 8; a pH value of about 8.5; a pH value ofabout 9; a pH value of about 9.5; a pH value of about 10; a pH value ofabout 11. It is understood that recitation of the above discrete valuesincludes a range between each recited value.

As another example, a buffer can have a pH value that is physiologicallyrelevant. A buffer can have a pH value of about 6 to about 8.

Kits

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to ascaffold, a modified simulated body fluid solution, or any other agentas described above. Such packaging of the components separately can, ifdesired, be presented in a pack or dispenser device which can containone or more unit dosage forms containing the composition. The pack can,for example, comprise metal or plastic foil such as a blister pack. Suchpackaging of the components separately can also, in certain instances,permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

All publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentdisclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1: Mineral Coating

The following example describes the mineralization of a polycaprolactone(PCL) scaffold.

A PCL scaffold was hydrolyzed in a 1.0 M NaOH solution for 60 minutes toproduce a hydrolyzed, carboxylic acid-rich surface. After hydrolysis ofthe PCL scaffold, a bone mineral coating was formed by immersion of aPCL scaffold in modified simulated body fluid (mSBF) and incubated.

The mSBF in this example was an inorganic solution having a similarcomposition to human plasma but with double the concentration of calciumand phosphate to enhance mineral growth, without organic components, atphysiological conditions, and continuous rotations. The mSBF solutionwas prepared according to the following. The following reagents wereadded to ddH₂O in the order shown: 141 mM NaCl, 4.0 mM KCl, 1.0 mMMgCl₂, 0.5 mM MgSO₄, 4.2 mM NaHCO₃, 20 mM Tris, 5 mM CaCl₂, and 2.0 mMKH₂PO₄. The solution was slowly heated to 37° C. and was adjusted to afinal pH of 6.8 using HCl and/or NaOH buffer solutions.

The incubation period was varied from 5 to 14 days. The mSBF solutionwas renewed every two days in order to maintain consistent ionicstrength throughout the coating process. The apatite (a group of calciumphosphates including bone mineral and the main inorganic constituent ofbones and teeth similar to hydroxyapatite) was nucleated and grown onthe surface of the PCL scaffold to form an integral part of the coatedPCL scaffold (see e.g., FIG. 1A-B).

The mechanism for mineral nucleation and growth on PCL is thought to bebased on the interaction of carboxylate ions and hydroxyl groups on thehydrolyzed PCL surface with calcium- and phosphate-rich nuclei in mSBFsolution. This mineral growth process is thought to mimic naturalbiomineralization processes and results in a mineral coating that issimilar in structure (plate-like nanostructure) and composition(carbonate-substituted, calcium-deficient hydroxyapatite phase) to humanbone mineral. Ca-deficient HA was measured by Ca/P ratio. Chemicals formSBF preparation were powder reagent grade chemicals. Water used was inaccordance with ISO3696:1987, grade 2.

After mineral coating, the scaffold was rinsed for 15 minutes in ddH₂Oto remove residual salts and was freeze dried overnight in a lyophilizerat a temperature of −40° C., under vacuum.

Example 2: Coating Specifications

The following example describes the coating specifications.

The assessment of mineral formation was performed by determining thechange in mass after coating compared with the initial mass (beforecoating).

Conditions were found to achieve a desired bone mineral coating onimplants (see e.g., TABLE 1).

TABLE 1 Parameters for bone mineral coating on implants CoatingSpecification (Coating alone) Property Description Analytical MethodChemical 97% Hydroxyapatite XRD composition and 3% Octacalciumconcentration (%) phosphate Ca/P ratio after heat 1.67 ≤ Ca/P ≤1.76 XRDtreatment at 1000° C., According to ISO 13779-2 Crystalline phaseHydroxyapatite XRD (2θ in the and octacalcium range of 15-35°, phosphatespecifically at 25.8°, 28.1°, 28.9°, 31.8°, and 32.1°) and FTIR (peaksin the 1400- 1500 cm⁻¹ region (for carbonate peaks) and 900-1100 cm⁻¹region (for phosphate peaks) % Crystallinity 96.5%* XRD Porosity (%)20-28%* SEM/Image J Pore size (nm) 100-350 nm* SEM/Image J Heavy metaltrace Arsenic (As) <2.1 ppm EDX for element element (ppm) as Cadmium(Cd) <0.1 ppm identification, ICP ref. in ISO 13779-3 Lead (Pb) <0.1 ppmfor element Mercury (Hg) <4.7 ppm concentrations TRS-SP0019-01 BoneMineral Coating, *Porosity and pore size studies of C-BVF

Example 3: Coating Dissolution

The following example describes coating dissolution rate in variousmedium conditions. It was shown that the phosphate buffer provideddecreased coating dissolution compared to Tris.

Under simulated physiological conditions, coating dissolution occurredwhen the environment was undersaturated with calcium and phosphate ions.This was the case for Tris-HCl. But, for the saline buffer, DPBS hadphosphate ions in the solution that were believed to be either blockingthe dissolution of phosphate ions from the coating or competing againsteach other for re-precipitation, resulting in the slower rate ofdissolution. A series of SEM micrographs (see e.g., FIG. 2) taken atdifferent time points and the amount of calcium (see e.g., FIG. 3) inthe release medium support the above hypothesis.

Example 4: Coating PCL Devices

The following example describes the details, proper instructions andsafety precautions for coating polycaprolactone (PCL) medical devicesvia modified simulated body fluid (mSBF) incubation, wherein the coatingprocedure applies to any size PCL medical device.

The modified simulated body fluid (mSBF) in this Example is a solutionwith a similar composition of human plasma but double the concentrationof calcium and phosphate. The mSBF solution described in this Example isused for incubating materials and nucleating an apatite-like mineral.

Materials.

Reagents and their respective desired concentration in final solution isas follows. Hydrolysis Reagents: NaOH (1.0 M). Coating Reagents: NaCl(141.0 mM); KCl (4.0 mM); MgSO₄ (0.5 mM); MgCl₂ (1.0 mM); NaHCO₃ (4.2mM); Tris(hydroxymethyl) aminomethane (20.2 mM); CaCl₂ (5.0 mM); KH₂PO₄(2.0 mM). Deionized ultra filtered (DIUF) water. Buffers: 2N HCl; 2NNaOH. Hot plate/stirrer; Magnetic stir bar; Spatula; Weighing paper orweigh boat; Analytical mass balance; pH meter; Glass beakers capable ofholding desired volume of mSBF; and 37° C. and 5% CO₂ Incubator.Rotating system: Labquake shaker system (for any container size up to175 mL); Large coating system (for coating large device, see e.g., TABLE2).

TABLE 2 Coating matrix using large coating system. Description ExampleSample size X ≥ 30 mm × 30 mm × Pig sleeve Degradation (dimensions in 50mm; Mass ≥3500 mg (80 × 45 × disc w. mm) 25 mm) handle (35 dia × 5 mm)Type of Custom made device for Large Large container large coatingsystem coating coating system system No. of 10-100, due to the large 1085 sample/ volume of mSBF container used we need to maximize the no. ofsample per coating duration Volume of Working volume of 3.0 gallons 2.75gallons mSBF (gallon) 1.5-5 gallons Frequency of 3 days  3 days  3 dayschanging mSBF (day) Coating 8-14 days, depending 10 days 8 days duration(day) on the size and no. of sample pH adjusting Daily Daily Daily

Hydrolysis Preparation.

Hydrolysis preparation includes determining the desired volume of 1MNaOH. See TABLE 3 for desired volume based on device size and weight.

TABLE 3 Hydrolysis matrix. Sample size 10 mm × 10 mm × 10 mm × 10 mm ×10 mm × 10 mm × X ≥25 mm × (dimensions in 10 mm ≤ X; 10 mm < X < 10 mm <X < 25 mm × 25 mm mm) and/or mass ≤250 mg 25 mm × 25 mm × 25 mm × 25 mm× mass ( mg) 5 mm; mass 10 mm; mass ≤2000 mg ≤3500 mg Type of 50 mLconical 50 mL conical 175 mL conical Depends on the container tube tubetube size of sample No. of 10-30, total 2-20, total 5-45, total Dependson the sample/ sample volume sample volume sample volume size of samplecontainer occupied ≤⅓ occupied ≤⅓ occupied ≤⅓ and selected total volumeof total volume of total volume of container container containercontainer Volume of 1M 50 mL 50 mL 150 mL Depends on the NaOH ( mL) sizeof sample and selected container Example Test cube Porous cylinderPorous cylinder Pig module (6 × 6 × 6 mm), (7 dia × 16 mm), (7 dia × 16mm), (35 × 30 × 10 mm), Monkey pin Human module Porous block Pig sleeve(10 × 5 × 3 mm), (20 × 20 × 12 mm), (25 × 25 × 6), (80 × 45 × 25 mm),Rabbit plate Monkey Monkey Human sleeve (10 × 5 × 5 mm) mandiblemandible (90 × 20 × 15 mm) (30 × 14 × 10 mm) (30 × 14 × 10 mm)

Determination of the mass of solid state NaOH is required to prepare a1M NaOH solution. Sample Calculation:

Desired volume of 1M NaOH: 400 mL

Required mass of solid state NaOH:

${m_{NaOH} = \lbrack{NaOH}\rbrack},{\frac{mol}{L}*Vol_{NaOH}},{{mL}*{MW}_{NaOH}}$$m_{NaOH} = {{\frac{1\mspace{14mu} {mol}}{L}*0.4\mspace{14mu} L*\frac{40\mspace{14mu} g}{mol}} = {16\mspace{14mu} g\mspace{14mu} {NaOH}}}$

Place hot plate/stirrer and analytical balance on rigid and leveledsurface.

Coating Preparation.

Determine the desired volume of mSBF. See TABLE 2 and TABLE 4 fordesired volume based on device size and weight. Note that for larger PCLdevices, the mSBF may need to be prepared in several batches; if so,determine the desired volume of the batch. Determine the mass of eachreagent needed to reach the desired concentration of each reagent (aslisted above) in the batch volume.

TABLE 4 Coating matrix. Sample size 10 mm × 10 mm × 10 mm × 10 mm × 10mm × 10 mm × (dimensions in mm) 10 mm ≤ X; mass 10 mm < X <25 mm × 10 mm< X <35 mm × and/or mass ( mg) ≤250 mg 25 mm × 90 mm; mass 35 mm × 50mm; mass ≤7500 mg ≤10,000 mg Type of container 15 mL conical tube 50 mLconical tube 175 mL conical tube No. of 1-10, total sample 1-15, totalsample 1-5, total sample sample/container volume occupied <½ volumeoccupied <½ volume occupied <½ total volume of total volume of totalvolume of container container container Volume of mSBF 15 mL 50 mL 175mL ( mL) Frequency of 2 days 2 days 2 days changing mSBF (days) Coatingduration 8 days 8 days 8 days (days) pH adjusting None None None ExampleTest cube Porous cylinder Pig module (6 × 6 × 6 mm), (7 dia × (35 × 30 ×10 mm), Monkey 16 mm), Human Disc pin (10 × 5 × 3 mm), module (35 dia ×5 mm) Rabbit plate (20 × 20 × 12 mm), (10 × 5 × 5 mm) Monkey mandible(30 × 14 × 10 mm), Human sleeve (90 × 20 × 15 mm)

Sample Calculation:

Desired volume of mSBF: 500 mL mSBF

Required mass of reagent #1 (e.g. NaCl, 141 mM)

${m_{NaCl} = \lbrack{NaCl}\rbrack},{\frac{mmol}{{ml}\;}*Vol_{mSBF}},{{ml}*{MW}_{NaCl}}$$m_{NaCl} = {{\frac{141 \times 10^{- 3}\mspace{14mu} {mmol}}{ml}*500\mspace{14mu} {ml}*\frac{58.4\mspace{14mu} {mg}}{m\; {mol}}} = {4120\mspace{14mu} {mg}}}$

Place hot plate/stirrer and analytical balance on rigid and leveledsurface. Check that the incubator is maintaining a 37° C. temperatureand a 5% CO₂ level. Check that the pH meter has been recentlycalibrated. If it has not been calibrated within the past 2 weeks,calibrate it according to the manufacturer's instructions.

Hydrolysis.

Measure the desired volume of DIUF water in an appropriately sizedbeaker. Place beaker on the hot plate/stirrer. Add magnetic stir bar tothe DIUF water. Set the stirrer between settings 5 and 10, depending onthe volume of DIUF water. Using a spatula and weighing paper or weighboat, weigh the required amount of NaOH on an analytical mass balance.Add the reagent to the beaker with the DIUF water. Stir continuouslyuntil completely dissolved. Add 1M NaOH to PCL devices. Hydrolyze thePCL devices in the 1M NaOH for 1 hour, rotating the container in acircular motion during hydrolysis. Make sure that the 1M NaOH covers theentire surface of the PCL device during hydrolysis. After hydrolysis iscomplete, remove 1M NaOH from the container with the PCL devices. Rinsethe PCL devices in DIUF water for 15 minutes. PCL devices ready forcoating.

Coating.

Measure the desired volume of DIUF water in an appropriately sizedbeaker. Place beaker on the hot plate/stirrer. Add magnetic stir bar tothe DIUF water. Set the stirrer between settings 5 and 10, depending onthe volume of DIUF water. Preheat the DIUF water by setting the hotplate to −40° C. (or setting 2-4). [Note: slowly heat the water fromroom temperature as reagents are added, and if 37° C. is reached, lowerthe heat setting to maintain the temperature of the water.] Using aspatula and weighing paper or weigh boat, weigh the required amount ofreagent #1 (NaCl) on an analytical mass balance. Add the reagent to thebeaker with the preheated DIUF water. While the solution is prepared,stir continuously. Wipe the spatula with ethanol or methanol after eachuse. Repeat for all of the reagents in order (as listed in Reagents).Add the reagents one at a time, ensuring that each reagent is fullydissolved before the next one is added. The temperature of the DIUFwater should be between 24-26° C. while reagents are being added, withthe DIUF water slowly heated to a temperature between 28-32° C. by thetime all reagents are added. Note: It is normal for the mSBF solution tobecome basic (pH ˜9) after addition of tris(hydroxymethyl) aminomethaneand to become cloudy after the addition of CaCl₂.

Measure the pH of the solution using the pH meter. Buffer the solutionto pH=6.8±0.1 using 2N HCl and 2N NaOH. Start buffering with 2N HCl asthe resulting solution is basic (pH 8-9). When the pH falls to ˜7.5, thesolution will start to become clear. If HCl is added in excess and thepH falls below 6.8±0.1, use NaOH to adjust the pH to 6.8±0.1. Whilebuffering the mSBF solution, continuously stir the solution and maintainthe temperature at 37° C. Slowly heat the solution to 37° C. (if thesolution has not yet reached 37° C. at the end of the bufferingprocess). Keep checking the pH and the temperature for stability. Repeatuntil the desired volume is reached. mSBF should be stored in the 37° C.incubator until use. Adjust the pH of the final bulk volume to 6.8±0.1.Add mSBF solution to PCL devices for coating. Incubate the PCL devicesin the 37° C. incubator, rotating the container in a circular motionduring incubation. Make sure that the mSBF covers the entire surface ofthe PCL device during incubation. See TABLE 2 and TABLE 4 for coatingduration, frequency of changing the mSBF solution, and pH adjustment (ifnecessary).

After incubation is complete, remove mSBF from the container with thePCL devices. Rinse the PCL devices in DIUF water for 15 minutes. Removethe DIUF water from the container with the PCL devices. Freeze the PCLdevices in a −20° C. freezer. Lyophilize the frozen PCL devices. PCLdevices ready for sterilization.

Special Precautions.

Wear latex gloves while handling all chemicals, solutions, and/orinstruments.

If the mSBF solution turns cloudy during incubation before 2 (or 3 daysfor large coating system set up), change the solution as soon aspossible.

Care and Maintenance of Equipment.

Daily: Calibrate the analytical mass balance to ensure accuracy ofweighed reagents. Weekly: Check the status of the CO₂ levels in theincubator. Replace CO₂ tank(s) as needed according to manufacturer'sinstructions. Every two weeks: Calibrate the pH meter according to themanufacturer's instructions.

Example 5: Surface Pre-Treatments of Titanium Sample Prior Coating

The following example describes the details, proper instructions, andsafety precautions for pretreating titanium samples.

Using 5 M NaOH. (a) Each step below need to be performed in a certifiedchemical hood and (b) volume of pre-treatment solution depends on sizeand shape of titanium samples, multiple samples may be pre-treated inthe same container.

Very slowly add NaOH pellets (Fisher Scientific Lot #115786) to stirringdeionized ultra-filtered (DIUF) water (Fisher Lot #140300) in a glassbeaker to make 5 M NaOH solution. Keep beaker on stirrer in hood withstirrer on ‘low’. Be mindful of fumes and rising temperatures—furtherslow down if excessive heating occurs. Let the solution stands forseveral minutes to cool down. Place Ti sample in clean glass beaker,pour NaOH solution in, cover the beaker with parafilm. Place beaker insonicator for 1 hr. Keep monitoring sample during sonicator process,take note for the volume of bubbles rising. Take Ti sample out, place itin another beaker with DIUF and leave on shaker for 15 min. Blot drywith paper towel, sample is now ready for coating process.

Using Piranha solution (conc. H₂SO₄+30% H₂O₂). Each step below isperformed in a certified chemical hood. Volume of pre-treatment solutiondepends on size and shape of titanium samples, multiple samples may bepre-treated in the same container. Add H₂SO₄ (Sigma Aldrich Lot#SHBB3339V) to a clean glass beaker in the hood. Very slowly add 30%H₂O₂ (Fisher Scientific Lot #102727) to H₂SO₄. The reaction can beexplosive and is highly exothermic, proceed very slowly and withcaution. Place Ti sample in clean glass beaker, carefully pour piranhasolution in. Monitor sample for 5-20 minutes; observe for volume ofbubbles rising. Immediately stop the reaction if there is any brownishcolor of sample/solution appear and take sample out.

Take Ti sample out quickly with forceps, rinse with copious amount ofDIUF, then place in DIUF on shaker for 15 min. Blot dry with papertowel, sample is now ready for coating process.

Coating Process.

After pre-treatment, each sample was coated for 4-6 days in coatingsolution (lx mSBF) at 37° C. with solution refreshed daily.

Example 6: Coating Enhances Osseointegration of Metal Surfaces

The unique nanoscale architecture of the coating may be applied tovirtually any underlying implant surface (e.g., plasma sprayedtitanium), reaching all pores of complex scaffolds and improving uponthe degree of osseointegration into micro-structured implants.

FIG. 4A and FIG. 4B show uncoated roughened titanium and coatedroughened titanium. Characteristics of the roughened titanium are a‘lava rock’ surface structure with a larger pore size between 200-525 μmand a smaller pore size in the range 25-65 μm. The average porevolume=60% and the average surface roughness=132 μm.

It was shown that the coating morphology and composition on titanium isconsistent with the same coating on PCL (see e.g., FIG. 8).

Example 7: Demineralized Bone Matrix (DBM) and Silver NanoparticlesIntegrated into Mineral Coated Scaffold

The following example describes the incorporation of demineralized bonematrix (DBM) to the mineral coating of the scaffold and silvernanoparticles integrated onto the mineral coating of scaffold.

DBM integrated with the mineral coating is presently thought to improvethe characteristics of the mineral-coated scaffold, such as theosteoinductive properties. DBM can be integrated into or onto themineral coating of the scaffold. DBM, such as demineralized bone extractor gel can be incorporated with the aqueous mineral coating solution inExample 4 to form a mixture of DBM and mineral coating solution to coata scaffold in a single coating step.

DBM can be coated onto a scaffold pre-coated with the mineral coating.The scaffold can be coated with DBM before, during, or after mineralcoating using the methods described herein.

Silver nanoparticles integrated with a scaffold or the mineral coatingare presently thought to provide biostatic, anti-infection properties tothe mineral-coated scaffold. Silver nanoparticles can be integrated ontothe mineral coating as described below. The nanoparticles can also beintegrated into the scaffold or coating before, during, or after DBMcoating using the methods described herein.

The following describes the incorporation of silver nanoparticles onto aCaP coated scaffold. CaP coated scaffolds are incubated in silvernitrate solution in deionized water (pH 7.0, 5 mL) to synthesize silverparticles on the surface or coating. In a set of experiments, CaPcoatings are pre-treated by incubating in citric acid solution indeionized water (pH 7.0, 5 mL) prior to silver nitrate incubation.

The concentrations of both solutions are varied (e.g., 1, 5, and 10 mM).Incubation times are also varied (e.g., 0.5, 1, and 4 hr). After citricacid pre-treatment, the resultant CaP coatings can be characterizedusing FT-IR, and crystallinity index can be calculated from Shemesh'smethod. The calcium released from citric acid-treated CaP coating in PBSat 37° C. can be quantified by colorimetric assay using Arsenazo III (MPBiochemicals, USA). The release medium can be collected at differenttimes to quantify calcium ions. Fresh PBS can be added for furtherincubation. The silver particles on CaP coatings can be imaged usingFE-SEM and elemental analysis using EDS. The silver particles scrapedfrom CaP coatings can be characterized using XRD.

The silver nanoparticle-integrated mineral-coated scaffold can befurther incorporated with DBM as described above. Alternatively, themineral coating can be incorporated with DBM before the addition ofsilver nanoparticles.

The DBM is integrated with a scaffold by mixing DBM with an aqueoussolution. The aqueous solution can be the mineral coating solution. Theaqueous solution can be a weak acid or guanidine hydrochloride. Themixture is constantly agitated for a set amount of time to produce anaqueous demineralized bone extract. The extract can then be filtered toremove any remaining solids, the acid neutralized or removed, and theextract used to coat the porous scaffold.

The DBM and aqueous solution or coating solution is mixed from about 8to 96 hours. The DBM and aqueous solution is mixed together withconstant agitation during that time. After mixing for the appropriateamount of time, the resulting demineralized bone extract can beseparated from any insoluble DBM remaining in the solution. The aqueoussolution or coating solution comprising the DBM is neutralized to a pHof from about 6.5 to about 7.5 by titration with an appropriatecounterion. The scaffold is then incubated according Example 4.

1. A method for producing a mineral coated scaffold comprising:contacting a scaffold comprising a matrix material and a modifiedsimulated body fluid; and incubating the scaffold and the modifiedsimulated body fluid for a period of time under conditions sufficient toform a mineral coated scaffold, wherein the mineral coating comprises acarbonate-substituted, calcium-deficient hydroxyapatite component,wherein the simulated body fluid is adjusted to a pH of about 6.5 toabout 7.2.
 2. The method of claim 1, wherein the matrix material is abiodegradable matrix material.
 3. The method of claim 1, wherein thematrix material comprises polycaprolactone (PCL), polyetheretherketone(PEEK), or titanium (Ti).
 4. The method of claim 1, further comprisingcombining NaCl, KCl, MgCl₂, MgSO₄, NaHCO₃, CaCl₂, and KH₂PO₄ to form themodified simulated body fluid.
 5. The method of claim 4, wherein: (i)NaCl has a concentration of about 100 mM to about 200 mM; (ii) KCl has aconcentration of about 1 mM to about 8 mM; (iii) MgCl₂ has aconcentration of about 0.2 mM to about 5 mM; (iv) MgSO₄ has aconcentration of about 0.2 mM to about 5 mM; (v) NaHCO₃ has aconcentration of about 1 mM to about 100 mM; (vi) CaCl₂ has aconcentration of about 2 mM to about 20 mM; and (vii) KH₂PO₄ has aconcentration of about 0.5 mM to about 10 mM.
 6. The method of claim 4,wherein: (i) NaCl has a concentration of about 141 mM; (ii) KCl has aconcentration of about 4.0 mM; (iii) MgCl₂ has a concentration of about1.0 mM; (iv) MgSO₄ has a concentration of about 0.5 mM; (v) NaHCO.sub.3has a concentration of about 4.2 mM; (vi) CaCl₂ has a concentration ofabout 5 mM; and (vii) KH₂PO₄ has a concentration of about 2.0 mM.
 7. Themethod of claim 1, wherein the modified simulated body fluid comprises abuffer.
 8. The method of claim 1, wherein the modified simulated bodyfluid comprises a buffer at a concentration of about 20 mM.
 9. Themethod of claim 1, wherein the modified simulated body fluid comprises abuffer selected from the group consisting of Tris-buffered saline, orPBS.
 10. The method of claim 1, wherein the period of time is from about5 days up to about 28 days.
 11. The method of claim 1, wherein theperiod of time is at least about 1 day; about 2 days; about 3 days;about 4 days; about 5 days; about 6 days; about 7 days; about 8 days;about 9 days; about 10 days; about 11 days; about 12 days; about 13days; about 14 days; about 15 days; about 16 days; about 17 days; about18 days; about 19 days; about 20 days; about 21 days; about 22 days;about 23 days; about 24 days; about 25 days; about 26 days; about 27days; or about 28 days.
 12. The method of claim 1, wherein the period oftime is about 5 days to about 14 days.
 13. The method of claim 1,further comprising hydrolyzing the scaffold.
 14. The method of claim 1,wherein the incubation comprises heating the modified simulated bodyfluid to physiologic temperature.
 15. The method of claim 14, whereinthe physiologic temperature is about 37° C. or the pH is about 6.8. 16.The method of claim 1, wherein incubating comprises replacing themodified simulated body fluid, replenishing the modified simulated bodyfluid, removing the modified simulated body fluid, or adding themodified simulated body fluid, wherein incubating comprises maintaininga concentration of modified simulated body fluid.
 17. The method ofclaim 17, wherein maintaining the concentration of modified simulatedbody fluid comprises replacing, replenishing, removing, or addingmodified simulated body fluid, NaCl, KCl, MgCl₂, MgSO₄, NaHCO₃, CaCl₂,or KH₂PO₄, or a combination thereof.
 18. The method of claim 1, whereinthe coating comprises about 97% hydroxyapatite.
 19. The method of claim1, wherein the coating comprises about 3% octacalcium phosphate.
 20. Themethod of claim 1, wherein the coating comprises between about 20% andabout 28% porosity.
 21. The method of claim 1, wherein the coatingcomprises between about 100 nm and about 350 nm pore diameter.
 22. Themethod of claim 1, wherein the coating comprises (i) about 1.37 to about1.61 Ca/P; or (ii) about 1.1 to about 1.3 Ca/P (calcium to phosphateratio).
 23. A mineral coated scaffold produced according to claim 1,comprising a scaffold comprising a matrix material, wherein the mineralcoating of the scaffold comprises a carbonate-substituted,calcium-deficient hydroxyapatite component.
 24. A mineral coatedscaffold comprising a matrix material, wherein the mineral coatingcomprises a carbonate-substituted, calcium-deficient hydroxyapatitecomponent; and wherein the mineral coating is formed by incubating thescaffold and a modified simulated body fluid, the modified simulatedbody fluid having a pH of about 6.5 to about 7.2 and the coatingcomprises at least one of silver particles or demineralized bone matrix.25. The mineral coated scaffold of claim 24, wherein the coatingcomprises: (i) about 97% hydroxyapatite; (ii) between about 20% andabout 28% porosity; (iii) between about 100 nm and about 350 nm porediameter; or (iv) about 1.1 to about 1.3 Ca/P; or about 1.37 to about1.61 Ca/P.
 26. The mineral coated scaffold of claim 24, wherein thecoating comprises octacalcium phosphate.
 27. the mineral coated scaffoldof claim 26, wherein the coating comprises 3% octacalcium phosphate.